Antibacterial aminoglycoside analogs

ABSTRACT

Compounds having antibacterial activity are disclosed. The compounds have the following structure (I): 
                         
including stereoisomers, pharmaceutically acceptable salts and prodrugs thereof, wherein Q 1 , Q 2 , Q 3 , R 1 , R 2  and R 3  are as defined herein. Methods associated with preparation and use of such compounds, as well as pharmaceutical compositions comprising such compounds, are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International PCT Application No.PCT/US2010/034888, filed May 14, 2010, now pending, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 61/178,826 filed May 15, 2009 and U.S. Provisional PatentApplication No. 61/312,353 filed Mar. 10, 2010. The foregoingapplications are incorporated herein by reference in their entireties.

BACKGROUND

1. Field

The present invention is directed to novel aminoglycoside compounds,more specifically, novel tobramycin derivatives, and methods for theirpreparation and use as therapeutic or prophylactic agents.

2. Description of the Related Art

A particular interest in modern drug discovery is the development ofnovel low molecular weight drugs that work by binding to RNA. RNA, whichserves as a messenger between DNA and proteins, was thought to be anentirely flexible molecule without significant structural complexity.Recent studies have revealed a surprising intricacy in RNA structure.RNA has a structural complexity rivaling proteins, rather than simplemotifs like DNA. Genome sequencing reveals both the sequences of theproteins and the mRNAs that encode them. Since proteins are synthesizedusing an RNA template, such proteins can be inhibited by preventingtheir production in the first place by interfering with the translationof the mRNA. Since both proteins and the RNAs are potential drugtargeting sites, the number of targets revealed from genome sequencingefforts is effectively doubled. These observations unlock a new world ofopportunities for the pharmaceutical industry to target RNA with smallmolecules.

Classical drug discovery has focused on proteins as targets forintervention. Proteins can be extremely difficult to isolate and purifyin the appropriate form for use in assays for drug screening. Manyproteins require post-translational modifications that occur only inspecific cell types under specific conditions. Proteins fold intoglobular domains with hydrophobic cores and hydrophilic and chargedgroups on the surface. Multiple subunits frequently form complexes,which may be required for a valid drug screen. Membrane proteins usuallyneed to be embedded in a membrane to retain their proper shape. Thesmallest practical unit of a protein that can be used in drug screeningis a globular domain. The notion of removing a single alpha helix orturn of a beta sheet and using it in a drug screen is not practical,since only the intact protein may have the appropriate 3-dimensionalshape for drug binding. Preparation of biologically active proteins forscreening is a major limitation in classical high throughput screening.Quite often the limiting reagent in high throughput screening efforts isa biologically active form of a protein which can also be quiteexpensive.

For screening to discover compounds that bind RNA targets, the classicapproaches used for proteins can be superceded with new approaches. AllRNAs are essentially equivalent in their solubility, ease of synthesisor use in assays. The physical properties of RNAs are independent of theprotein they encode. They may be readily prepared in large quantitythrough either chemical or enzymatic synthesis and are not extensivelymodified in vivo. With RNA, the smallest practical unit for drug bindingis the functional subdomain. A functional subdomain in RNA is a fragmentthat, when removed from the larger RNA and studied in isolation, retainsits biologically relevant shape and protein or RNA-binding properties.The size and composition of RNA functional subdomains make themaccessible by enzymatic or chemical synthesis. The structural biologycommunity has developed significant experience in identification offunctional RNA subdomains in order to facilitate structural studies bytechniques such as NMR spectroscopy. For example, small analogs of thedecoding region of 16S rRNA (the A-site) have been identified ascontaining only the essential region, and have been shown to bindantibiotics in the same fashion as the intact ribosome.

The binding sites on RNA are hydrophilic and relatively open as comparedto proteins. The potential for small molecule recognition based on shapeis enhanced by the deformability of RNA. The binding of molecules tospecific RNA targets can be determined by global conformation and thedistribution of charged, aromatic, and hydrogen bonding groups off of arelatively rigid scaffold. Properly placed positive charges are believedto be important, since long-range electrostatic interactions can be usedto steer molecules into a binding pocket with the proper orientation. Instructures where nucleobases are exposed, stacking interactions witharomatic functional groups may contribute to the binding interaction.The major groove of RNA provides many sites for specific hydrogenbonding with a ligand. These include the aromatic N7 nitrogen atoms ofadenosine and guanosine, the O4 and O6 oxygen atoms of uridine andguanosine, and the amines of adenosine and cytidine. The rich structuraland sequence diversity of RNA suggests to us that ligands can be createdwith high affinity and specificity for their target.

Although our understanding of RNA structure and folding, as well as themodes in which RNA is recognized by other ligands, is far from beingcomprehensive, significant progress has been made in the last decade(see, e.g., Chow, C. S.; Bogdan, F. M., Chem. Rev., 1997, 97, 1489 andWallis, M. G.; Schroeder, R., Prog. Biophys. Molec. Biol. 1997, 67,141). Despite the central role RNA plays in the replication of bacteria,drugs that target these pivotal RNA sites of these pathogens are scarce.The increasing problem of bacterial resistance to antibiotics makes thesearch for novel RNA binders of crucial importance.

Certain small molecules can bind and block essential functions of RNA.Examples of such molecules include the aminoglycoside antibiotics anddrugs such as erythromycin which binds to bacterial rRNA and releasespeptidyl-tRNA and mRNA. Aminoglycoside antibiotics have long been knownto bind RNA. They exert their antibacterial effects by binding tospecific target sites in the bacterial ribosome. For the structurallyrelated antibiotics neamine, ribostamycin, neomycin B, and paromomycin,the binding site has been localized to the A-site of the prokaryotic 16Sribosomal decoding region RNA (see Moazed, D.; Noller, H. F., Nature,1987, 327, 389). Binding of aminoglycosides to this RNA targetinterferes with the fidelity of mRNA translation and results inmiscoding and truncation, leading ultimately to bacterial cell death(see Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C., J. Am. Chem. Soc.,1998, 120, 1965).

There is a need in the art for new chemical entities that work againstbacteria with broad-spectrum activity. Perhaps the biggest challenge indiscovering RNA-binding antibacterial drugs is identifying vitalstructures common to bacteria that can be disabled by small moleculedrug binding. A challenge in targeting RNA with small molecules is todevelop a chemical strategy which recognizes specific shapes of RNA.There are three sets of data that provide hints on how to do this:natural protein interactions with RNA, natural product antibiotics thatbind RNA, and man-made RNAs (aptamers) that bind proteins and othermolecules. Each data set, however, provides different insights to theproblem.

Several classes of drugs obtained from natural sources have been shownto work by binding to RNA or RNA/protein complexes. These include threedifferent structural classes of antibiotics: thiostreptone, theaminoglycoside family and the macrolide family of antibiotics. Theseexamples provide powerful clues to how small molecules and targets mightbe selected. Nature has selected RNA targets in the ribosome, one of themost ancient and conserved targets in bacteria. Since antibacterialdrugs are desired to be potent and have broad-spectrum activity, theseancient processes, fundamental to all bacterial life, representattractive targets. The closer we get to ancient conserved functions themore likely we are to find broadly conserved RNA shapes. It is importantto also consider the shape of the equivalent structure in humans, sincebacteria were unlikely to have considered the therapeutic index of theirRNAs while evolving them.

A large number of natural antibiotics exist, these include theaminoglycosides, such as, kirromycin, neomycin, paromomycin,thiostrepton, and many others. They are very potent, bactericidalcompounds that bind RNA of the small ribosomal subunit. The bactericidalaction is mediated by binding to the bacterial RNA in a fashion thatleads to misreading of the genetic code. Misreading of the code duringtranslation of integral membrane proteins is thought to produce abnormalproteins that compromise the barrier properties of the bacterialmembrane.

Antibiotics are chemical substances produced by various species ofmicroorganisms (bacteria, fungi, actinomycetes) that suppress the growthof other microorganisms and may eventually destroy them. However, commonusage often extends the term antibiotics to include syntheticantibacterial agents, such as the sulfonamides, and quinolines, that arenot products of microbes. The number of antibiotics that have beenidentified now extends into the hundreds, and many of these have beendeveloped to the stage where they are of value in the therapy ofinfectious diseases. Antibiotics differ markedly in physical, chemical,and pharmacological properties, antibacterial spectra, and mechanisms ofaction. In recent years, knowledge of molecular mechanisms of bacterial,fungal, and viral replication has greatly facilitated rationaldevelopment of compounds that can interfere with the life cycles ofthese microorganisms.

At least 30% of all hospitalized patients now receive one or morecourses of therapy with antibiotics, and millions of potentially fatalinfections have been cured. At the same time, these pharmaceuticalagents have become among the most misused of those available to thepracticing physician. One result of widespread use of antimicrobialagents has been the emergence of antibiotic-resistant pathogens, whichin turn has created an ever-increasing need for new drugs. Many of theseagents have also contributed significantly to the rising costs ofmedical care.

When the antimicrobial activity of a new agent is first tested, apattern of sensitivity and resistance is usually defined. Unfortunately,this spectrum of activity can subsequently change to a remarkabledegree, because microorganisms have evolved the array of ingeniousalterations discussed above that allow them to survive in the presenceof antibiotics. The mechanism of drug resistance varies frommicroorganism to microorganism and from drug to drug.

The development of resistance to antibiotics usually involves a stablegenetic change, inheritable from generation to generation. Any of themechanisms that result in alteration of bacterial genetic compositioncan operate. While mutation is frequently the cause, resistance toantimicrobial agents may be acquired through transfer of geneticmaterial from one bacterium to another by transduction, transformationor conjugation.

For the foregoing reasons, while progress has been made in this field,there is a need for new chemical entities that possess antibacterialactivity. Further, in order to accelerate the drug discovery process,new methods for synthesizing aminoglycoside antibiotics are needed toprovide an array of compounds that are potentially new drugs for thetreatment of bacterial infections. The present invention fulfills theseneeds and provides further related advantages.

BRIEF SUMMARY

In brief, the present invention is directed to novel aminoglycosidecompounds, more specifically, novel tobramycin derivatives, havingantibacterial activity, including stereoisomers, pharmaceuticallyacceptable salts and prodrugs thereof, and the use of such compounds inthe treatment of bacterial infections.

In one embodiment, compounds having the following structure (I) areprovided:

or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof,

wherein:

Q₁ is optionally substituted alkyl,

Q₂ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

Q₃ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

each R₁ and R₂ is, independently, hydrogen or an amino protecting group;

each R₃ is, independently, hydrogen or a hydroxyl protecting group;

each R₄, R₅, R₇ and R₈ is, independently, hydrogen or C₁-C₆ alkyloptionally substituted with one or more halogen, hydroxyl or amino;

each R₆ is, independently, hydrogen, halogen, hydroxyl, amino or C₁-C₆alkyl;

or R₄ and R₅ together with the atoms to which they are attached can forma heterocyclic ring having from 4 to 6 ring atoms, or R₅ and one R₆together with the atoms to which they are attached can form aheterocyclic ring having from 3 to 6 ring atoms, or R₄ and one R₆together with the atoms to which they are attached can form acarbocyclic ring having from 3 to 6 ring atoms, or R₇ and R₈ togetherwith the atom to which they are attached can form a heterocyclic ringhaving from 3 to 6 ring atoms;

each R₉ is, independently, hydrogen, hydroxyl, amino or C₁-C₆ alkyloptionally substituted with one or more halogen, hydroxyl or amino;

each R₁₀ is, independently, hydrogen, halogen, hydroxyl, amino or C₁-C₆alkyl;

or R₉ and one R₁₀ together with the atoms to which they are attached canform a heterocyclic ring having from 3 to 6 ring atoms; and

n is an integer from 0 to 4, and

wherein (i) at least one of Q₂ and Q₃ are other than hydrogen, (ii) ifQ₁ is —C(═O)CH(OH)(CH₂)₂NH₂, then Q₂ is not methyl, and (iii) Q₁, Q₂ andQ₃ are not all —C(═O)CH₃.

In another embodiment, a pharmaceutical composition is providedcomprising a compound having structure (I), or a stereoisomer,pharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, a method of using a compound having structure (I)in therapy is provided. In particular, the present invention provides amethod of treating a bacterial infection in a mammal comprisingadministering to the mammal an effective amount of a compound havingstructure (I), or a stereoisomer, pharmaceutically acceptable salt orprodrug thereof.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

As used in the specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated.

“Amino” refers to the —NH₂ radical.

“Cyano” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. Unless stated otherwise specifically in the specification, analkyl group may be optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain may be optionally substituted.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup may be optionally substituted.

“Alkylamino” refers to a radical of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl radical as defined abovecontaining one to twelve carbon atoms. Unless stated otherwisespecifically in the specification, an alkylamino group may be optionallysubstituted.

“Thioalkyl” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, a thioalkylgroup may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 18 carbon atoms and at least one aromatic ring. For purposes ofthis invention, the aryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems. Aryl radicals include, but are not limited to, arylradicals derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s-indacene, indane, indene, naphthalene, phenalene,phenanthrene, pleiadene, pyrene, and triphenylene. Unless statedotherwise specifically in the specification, the term “aryl” or theprefix “ar-” (such as in “aralkyl”) is meant to include aryl radicalsthat are optionally substituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl and thelike. Unless stated otherwise specifically in the specification, anaralkyl group may be optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group may be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —R_(b)R_(d) whereR_(d) is an alkylene chain as defined above and R_(g) is a cycloalkylradical as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the invention. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring radical which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“N-heterocyclyl” refers to a heterocyclyl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl radical to the rest of the molecule is through anitrogen atom in the heterocyclyl radical. Unless stated otherwisespecifically in the specification, a N-heterocyclyl group may beoptionally substituted.

“Heterocyclylalkyl” refers to a radical of the formula —R_(b)R_(e) whereR_(b) is an alkylene chain as defined above and R_(e) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. Unless stated otherwisespecifically in the specification, a heterocyclylalkyl group may beoptionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and at least one aromatic ring. For purposes of this invention,the heteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

“N-heteroaryl” refers to a heteroaryl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. Unless stated otherwise specifically inthe specification, an N-heteroaryl group may be optionally substituted.

“Heteroarylalkyl” refers to a radical of the formula —R_(b)R_(f) whereR_(b) is an alkylene chain as defined above and R_(f) is a heteroarylradical as defined above. Unless stated otherwise specifically in thespecification, a heteroarylalkyl group may be optionally substituted.

The term “substituted” used herein means any of the above groups (i.e.,alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl)wherein at least one hydrogen atom is replaced by a bond to anon-hydrogen atoms such as, but not limited to: a halogen atom such asF, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups,alkoxy groups, and ester groups; a sulfur atom in groups such as thiolgroups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxidegroups; a nitrogen atom in groups such as amines, amides, alkylamines,dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides,imides, and enamines; a silicon atom in groups such as trialkylsilylgroups, dialkylarylsilyl groups, alkyldiarylsilyl groups, andtriarylsilyl groups; and other heteroatoms in various other groups.“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced by a higher-order bond (e.g., a double- ortriple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl,and ester groups; and nitrogen in groups such as imines, oximes,hydrazones, and nitriles. For example, “substituted” includes any of theabove groups in which one or more hydrogen atoms are replaced with—NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)C(═NR_(g))NR_(g)R_(h), —NR_(g)SO₂R_(h),—OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g),—SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h). “Substituted also means anyof the above groups in which one or more hydrogen atoms are replacedwith —C(═O)R_(g), —C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g),—CH₂SO₂NR_(g)R_(h). In the foregoing, R_(g) and R_(h) are the same ordifferent and independently hydrogen, alkyl, alkoxy, alkylamino,thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl,heterocyclyl. N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl. “Substituted” further means any ofthe above groups in which one or more hydrogen atoms are replaced by abond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo,alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkylgroup. In addition, each of the foregoing substituents may also beoptionally substituted with one or more of the above substituents.

The term “protecting group,” as used herein, refers to a labile chemicalmoiety which is known in the art to protect reactive groups includingwithout limitation, hydroxyl and amino groups, against undesiredreactions during synthetic procedures. Hydroxyl and amino groups whichprotected with a protecting group are referred to herein as “protectedhydroxyl groups” and “protected amino groups”, respectively. Protectinggroups are typically used selectively and/or orthogonally to protectsites during reactions at other reactive sites and can then be removedto leave the unprotected group as is or available for further reactions.Protecting groups as known in the art are described generally in Greeneand Wuts, Protective Groups in Organic Synthesis, 3rd edition, JohnWiley & Sons, New York (1999). Groups can be selectively incorporatedinto aminoglycosides of the invention as precursors. For example anamino group can be placed into a compound of the invention as an azidogroup that can be chemically converted to the amino group at a desiredpoint in the synthesis. Generally, groups are protected or present as aprecursor that will be inert to reactions that modify other areas of theparent molecule for conversion into their final groups at an appropriatetime. Further representative protecting or precursor groups arediscussed in Agrawal, et al., Protocols for Oligonucleotide Conjugates,Eds, Humana Press; New Jersey, 1994; Vol. 26 pp. 1-72. Examples of“hydroxyl protecting groups” include, but are not limited to, t-butyl,t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,benzoylformate, acetate, chloroacetate, trichloroacetate,trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate,9-fluorenylmethyl carbonate, mesylate and tosylate. Examples of “aminoprotecting groups” include, but are not limited to, carbamate-protectinggroups, such as 2-trimethylsilylethoxycarbonyl (Teoc),1-methyl-1-(4-biphenylyl)ethoxy-carbonyl (Bpoc), t-butoxycarbonyl (BOC),allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), andbenzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imineand cyclic imide protecting groups, such as phthalimido anddithiasuccinoyl.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi.T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxyl, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxyl,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass allpharmaceutically acceptable compounds of structure (I) beingisotopically-labelled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²²I, and ¹²⁵I, respectively. These radiolabelledcompounds could be useful to help determine or measure the effectivenessof the compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled compounds of structure (I), forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies. The radioactive isotopestritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful forthis purpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed in the Preparations and Examples as set out below using anappropriate isotopically-labeled reagent in place of the non-labeledreagent previously employed.

The invention disclosed herein is also meant to encompass the in vivometabolic products of the disclosed compounds. Such products may resultfrom, for example, the oxidation, reduction, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising administering a compound of thisinvention to a mammal for a period of time sufficient to yield ametabolic product thereof. Such products are typically identified byadministering a radiolabelled compound of the invention in a detectabledose to an animal, such as rat, mouse, guinea pig, monkey, or to human,allowing sufficient time for metabolism to occur, and isolating itsconversion products from the urine, blood or other biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the invention may be truesolvates, while in other cases, the compound of the invention may merelyretain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the invention which, when administered to amammal, preferably a human, is sufficient to effect treatment, asdefined below, of a bacterial infection in the mammal, preferably ahuman. The amount of a compound of the invention which constitutes a“therapeutically effective amount” will vary depending on the compound,the condition and its severity, the manner of administration, and theage of the mammal to be treated, but can be determined routinely by oneof ordinary skill in the art having regard to his own knowledge and tothis disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds of the invention, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centres of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

As noted above, in one embodiment of the present invention, compoundshaving antibacterial activity are provided, the compounds having thefollowing structure (I):

or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof,

wherein:

Q₁ is optionally substituted alkyl,

Q₂ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

Q₃ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

each R₁ and R₂ is, independently, hydrogen or an amino protecting group;

each R₃ is, independently, hydrogen or a hydroxyl protecting group;

each R₄, R₅, R₇ and R₈ is, independently, hydrogen or C₁-C₆ alkyloptionally substituted with one or more halogen, hydroxyl or amino;

each R₆ is, independently, hydrogen, halogen, hydroxyl, amino or C₁-C₆alkyl;

or R₄ and R₅ together with the atoms to which they are attached can forma heterocyclic ring having from 4 to 6 ring atoms, or R₅ and one R₆together with the atoms to which they are attached can form aheterocyclic ring having from 3 to 6 ring atoms, or R₄ and one R₆together with the atoms to which they are attached can form acarbocyclic ring having from 3 to 6 ring atoms, or R₇ and R₈ togetherwith the atom to which they are attached can form a heterocyclic ringhaving from 3 to 6 ring atoms;

each R₉ is, independently, hydrogen, hydroxyl, amino or C₁-C₆ alkyloptionally substituted with one or more halogen, hydroxyl or amino;

each R₁₀ is, independently, hydrogen, halogen, hydroxyl, amino or C₁-C₆alkyl;

or R₉ and one R₁₀ together with the atoms to which they are attached canform a heterocyclic ring having from 3 to 6 ring atoms; and

n is an integer from 0 to 4, and

wherein (i) at least one of Q₂ and Q₃ are other than hydrogen, (ii) ifQ₁ is —C(═O)CH(OH)(CH₂)₂NH₂, then Q₂ is not methyl, and (iii) Q₁, Q₂ andQ₃ are not all —C(═O)CH₃.

In further embodiments,

Q₁ is optionally substituted alkyl, —C(═O)—H,

Q₂ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

Q₃ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

each R₁ and R₂ is, independently, hydrogen or an amino protecting group;

each R₃ is, independently, hydrogen or a hydroxyl protecting group;

each R₄, R₅, R₆, R₇ and R₈ is, independently, hydrogen or C₁-C₆ alkyl,or R₄ and R₅ together with the atoms to which they are attached can forma heterocyclic ring having from 4 to 6 ring atoms, or R₅ and R₆ togetherwith the atoms to which they are attached can form a heterocyclic ringhaving from 4 to 6 ring atoms, or R₄ and R₆ together with the atoms towhich they are attached can form a carbocyclic ring having from 3 to 6ring atoms, or R₇ and R₈ together with the atom to which they areattached can form a heterocyclic ring having from 4 to 6 ring atoms;

each R₉ and R₁₀ is, independently, hydrogen, hydroxyl, amino or C₁-C₆alkyl, or R₉ and R₁₀ together with the atoms to which they are attachedcan form a heterocyclic ring having from 4 to 6 ring atoms;

each n is, independently, an integer from 0 to 4; and

each m is, independently, an integer from 0 to 4, and

wherein (i) at least one of Q₂ and Q₃ are other than hydrogen, (ii) ifQ₁ is —C(═O)CH(OH)(CH₂)₂NH₂, then Q₂ is not methyl, and (iii) Q₁, Q₂ andQ₃ are not all —C(═O)CH₃.

In other further embodiments, Q₁ is:

wherein: R₄ is hydrogen; R₅ is hydrogen; and n is an integer from 1 to4. In further embodiments, each R₆ is hydrogen. For example, in morespecific embodiments of the foregoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein: R₄ is hydrogen; R₅ and one R₆ together with the atoms to whichthey are attached form a heterocyclic ring having from 3 to 6 ringatoms; and n is an integer from 1 to 4. For example, in more specificembodiments of the foregoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein: R₄ and R₅ together with the atoms to which they are attachedform a heterocyclic ring having from 4 to 6 ring atoms; and n is aninteger from 1 to 4. In further embodiments, each R₆ is hydrogen. Forexample, in more specific embodiments of the foregoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein: R₅ is hydrogen; R₄ and one R₆ together with the atoms to whichthey are attached form a carbocyclic ring having from 3 to 6 ring atoms;and n is an integer from 1 to 4. For example, in more specificembodiments of the foregoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein: R₄ is hydrogen; R₇ is hydrogen; R_(S) is hydrogen; and n is aninteger from 1 to 4. In further embodiments, each R₆ is hydrogen. Forexample, in more specific embodiments of the foregoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein: R₄ and one R₆ together with the atoms to which they areattached form a carbocyclic ring having from 3 to 6 ring atoms; R₇ ishydrogen; R₈ is hydrogen; and n is an integer from 1 to 4. For example,in more specific embodiments of the foregoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein R₅ is hydrogen. In further embodiments, each R₆ is hydrogen. Forexample, in more specific embodiments of the foregoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein: R₇ is hydrogen; and R₈ is hydrogen. In further embodiments,each R₆ is hydrogen. For example, in more specific embodiments of theforegoing, Q₁ is:

In other further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein R₅ is hydrogen. In further embodiments, each R₆ is hydrogen. Inother further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein: R₇ is hydrogen; and R₈ is hydrogen. In further embodiments,each R₆ is hydrogen. In other further embodiments, at least one R₆ ishalogen.

In other further embodiments, Q₁ is:

wherein R₅ is hydrogen. In further embodiments, each R₆ is hydrogen. Inother further embodiments, at least one R₆ is halogen.

In other further embodiments, Q₁ is:

wherein R₉ is hydrogen. In further embodiments, each R₁₀ is hydrogen. Inother further embodiments, at least one R₁₀ is halogen.

In other further embodiments, Q₁ is:

wherein: R₇ is hydrogen; and R₈ is hydrogen. In further embodiments,each R₁₀ is hydrogen. In other further embodiments, at least one R₁₀ ishalogen.

In other further embodiments, Q₁ is:

wherein R₄ is hydrogen. In further embodiments, each R₆ is hydrogen. Inother further embodiments, at least one R₆ is halogen. In other furtherembodiments, Q₁ is —C(═O)H.

In other further embodiments, Q₁ is optionally substituted alkyl. Forexample, in more specific embodiments of the foregoing, Q₁ isunsubstituted or Q₁ is substituted with one or more halogen, hydroxyl oramino.

In other further embodiments, Q₂ is other than hydrogen.

In other further embodiments, Q₂ is optionally substituted alkyl. Forexample, in more specific embodiments, Q₂ is unsubstituted. In othermore specific embodiments, Q₂ is substituted with hydroxyl or amino.

In other further embodiments, Q₂ is optionally substituted cycloalkyl.For example, in more specific embodiments, Q₂ is unsubstituted. In othermore specific embodiments, Q₂ is substituted with hydroxyl or amino.

In other further embodiments, Q₂ is optionally substitutedcycloalkylalkyl. For example, in more specific embodiments, Q₂ isunsubstituted. In other more specific embodiments, Q₂ is substitutedwith hydroxyl or amino.

In other further embodiments, Q₂ is optionally substituted heterocyclyl.For example, in more specific embodiments, Q₂ is unsubstituted. In othermore specific embodiments, Q₂ is substituted with hydroxyl or amino.

In other further embodiments, Q₂ is optionally substitutedheterocyclylalkyl. For example, in more specific embodiments, Q₂ isunsubstituted. In other more specific embodiments, Q₂ is substitutedwith hydroxyl or amino.

In other further embodiments, Q₂ is hydrogen.

In other further embodiments, Q₃ is other than hydrogen.

In other further embodiments, Q₃ is optionally substituted alkyl. Forexample, in more specific embodiments, Q₃ is unsubstituted. In othermore specific embodiments, Q₃ is substituted with hydroxyl or amino.

In other further embodiments, Q₃ is optionally substituted cycloalkyl.For example, in more specific embodiments, Q₃ is unsubstituted. In othermore specific embodiments, Q₃ is substituted with hydroxyl or amino.

In other further embodiments, Q₃ is optionally substitutedcycloalkylalkyl. For example, in more specific embodiments, Q₃ isunsubstituted. In other more specific embodiments, Q₃ is substitutedwith hydroxyl or amino.

In other further embodiments, Q₃ is optionally substituted heterocyclyl.For example, in more specific embodiments, Q₃ is unsubstituted. In othermore specific embodiments, Q₃ is substituted with hydroxyl or amino.

In other further embodiments, Q₃ is optionally substitutedheterocyclylalkyl. For example, in more specific embodiments, Q₃ isunsubstituted. In other more specific embodiments, Q₃ is substitutedwith hydroxyl or amino.

In other further embodiments, Q₃ is —C(═NH)NH₂.

In other further embodiments, Q₃ is hydrogen.

In other further embodiments, each R₁, R₂ and R₃ is hydrogen.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific substituent set forth herein for aQ₁, Q₂, Q₃, R₁, R₂ or R₃ group in the compounds of structure (I), as setforth above, may be independently combined with other embodiments and/orsubstituents of compounds of structure (I) to form embodiments of theinvention not specifically set forth above. In addition, in the eventthat a list of substitutents is listed for any particular substituentgroup in a particular embodiment and/or claim, it is understood thateach individual substituent may be deleted from the particularembodiment and/or claim and that the remaining list of substituents willbe considered to be within the scope of the invention.

For the purposes of administration, the compounds of the presentinvention may be administered as a raw chemical or may be formulated aspharmaceutical compositions. Pharmaceutical compositions of the presentinvention comprise a compound of structure (I) and a pharmaceuticallyacceptable carrier, diluent or excipient. The compound of structure (I)is present in the composition in an amount which is effective to treat aparticular disease or condition of interest—that is, in an amountsufficient to treat a bacterial infection, and preferably withacceptable toxicity to the patient. The antibacterial activity ofcompounds of structure (I) can be determined by one skilled in the art,for example, as described in the Examples below. Appropriateconcentrations and dosages can be readily determined by one skilled inthe art.

The compounds of the present invention possess antibacterial activityagainst a wide spectrum of gram positive and gram negative bacteria, aswell as enterobacteria and anaerobes. Representative susceptibleorganisms generally include those gram positive and gram negative,aerobic and anaerobic organisms whose growth can be inhibited by thecompounds of the invention such as Staphylococcus, Lactobacillus,Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella,Pseudomonas, Acinetobacter, Mycobacterium, Proteus, Campylobacter,Citrobacter, Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium,Salmonella, Shigella, Serratia, Haemophilus, Brucella, Francisella,Anthracis, Yersinia, Corynebacterium, Moraxella, Enterococcus, and otherorganisms.

Administration of the compounds of the invention, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of administration of agents for serving similar utilities. Thepharmaceutical compositions of the invention can be prepared bycombining a compound of the invention with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Typical routes of administering such pharmaceuticalcompositions include, without limitation, oral, topical, transdermal,inhalation, parenteral, sublingual, buccal, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. Pharmaceutical compositions of the invention areformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that will be administered to a subject or patient take theform of one or more dosage units, where for example, a tablet may be asingle dosage unit, and a container of a compound of the invention inaerosol form may hold a plurality of dosage units. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington: The Science andPractice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy andScience, 2000). The composition to be administered will, in any event,contain a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable salt thereof, for treatmentof a disease or condition of interest in accordance with the teachingsof this invention.

A pharmaceutical composition of the invention may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for eitherparenteral or oral administration should contain an amount of a compoundof the invention such that a suitable dosage will be obtained.

The pharmaceutical composition of the invention may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

The pharmaceutical composition of the invention may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of the invention may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the invention in solid or liquid formmay include an agent that binds to the compound of the invention andthereby assists in the delivery of the compound. Suitable agents thatmay act in this capacity include a monoclonal or polyclonal antibody, aprotein or a liposome.

The pharmaceutical composition of the invention may consist of dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of the invention may bedelivered in single phase, bi-phasic, or tri-phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols.

The pharmaceutical compositions of the invention may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining a compound of the invention with sterile,distilled water so as to form a solution. A surfactant may be added tofacilitate the formation of a homogeneous solution or suspension.Surfactants are compounds that non-covalently interact with the compoundof the invention so as to facilitate dissolution or homogeneoussuspension of the compound in the aqueous delivery system,

The compounds of the invention, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific compound employed; the metabolic stability and length ofaction of the compound; the age, body weight, general health, sex, anddiet of the patient; the mode and time of administration; the rate ofexcretion; the drug combination; the severity of the particular disorderor condition; and the subject undergoing therapy.

Compounds of the invention, or pharmaceutically acceptable derivativesthereof, may also be administered simultaneously with, prior to, orafter administration of one or more other therapeutic agents. Suchcombination therapy includes administration of a single pharmaceuticaldosage formulation which contains a compound of the invention and one ormore additional active agents, as well as administration of the compoundof the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, a compound of theinvention and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof the invention and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in thesynthetic processes described herein the functional groups ofintermediate compounds may need to be protected by suitable protectinggroups. Such functional groups include hydroxyl, amino, mercapto andcarboxylic acid. Suitable protecting groups for hydroxyl includetrialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl,t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, andthe like. Suitable protecting groups for amino, amidino and guanidinoinclude t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitableprotecting groups for mercapto include —C(O)—R″ (where R″ is alkyl, arylor arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although aprotected derivative of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of theinvention can be converted to their free base or acid form by standardtechniques.

The following Examples illustrate various methods of making compounds ofthis invention, i.e., compounds of structure (I):

wherein Q₁, Q₂, Q₃, R₁, R₂ and R₃ are as defined herein. It isunderstood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othercompounds of structure (I) not specifically illustrated below by usingthe appropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,5th edition (Wiley, December 2000)) or prepared as described herein.

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES

Example 1

Example 2

Example 3

Example 4

Example 5

Example 6

Example 7

Example 8

Example 9

Example 10

Example 11

Example 12

Example 13

Example 14

Representative Coupling Agents

As one of skill in the art will appreciate, other representativecoupling agents that may be utilized in the above examples include, butare not limited to, the following.

General Synthetic Procedures

Procedure 1: Reductive Amination

Method A: To a stirring solution of the aminoglycoside derivative (0.06mmol) in MeOH (2 mL) was added the aldehyde (0.068 mmol), silicasupported cyanoborohydride (0.1 g, 1.0 mmol/g), and the reaction mixturewas heated by microwave irradiation to 100° C. (100 watts power) for 15minutes. The reaction was checked by MS for completeness, and oncecomplete all solvent was removed by rotary evaporation. The resultingresidue was dissolved in EtOAc (20 ml), and washed with 5% NaHCO₃ (2×5mL), followed by brine (5 mL). The organic phase was then dried overNa₂SO₄, filtered and the solvent was removed by rotary evaporation.

Method B: To a solution of aminoglycoside derivative (0.078 mmol) in DMF(1 ml) were added 3 Å molecular sieves (15-20), followed by the aldehyde(0.15 mmol) and the reaction was shaken for 2.5 hours. The reaction waschecked by MS for completeness and, if needed, more aldehyde (0.5 eq)was added. The reaction mixture was then added dropwise to a stirringsolution of NaBH₄ (0.78 mmol) in MeOH (2 mL) at 0° C., and the reactionwas stirred for 1 hour. The reaction was diluted with H₂O (2 mL) andEtOAc (2 ml). The organic layer was separated and the aqueous layer wasextracted with EtOAc (3×3 mL). The combined organic layers were driedover Na₂SO₄, filtered and concentrated to dryness.

Procedure 2: PNZ Deprotection

To a stirring solution of the PNZ protected aminoglycoside derivative(0.054 mmol) in EtOH (1.5 mL) and H₂O (1 mL) was added 1N NaOH (0.3 mL),followed by Na₂S₂O₄ (0.315 mmol), and the reaction mixture was heated at70° C. for 12 hours. The reaction progress was monitored by MS. Oncecomplete, the reaction mixture was diluted with H₂O (5 mL) and thenextracted with EtOAc (2×10 mL). The combined organic layers were washedwith H₂O (2×5 mL), brine (5 mL), dried over Na₂SO₄, filtered andconcentrated to dryness.

Procedure 3: Boc Deprotection (Tert-Butyl Dimethyl Silyl ProtectingGroup is Removed Under these Conditions)

Important: Before Boc deprotection a sample must be dried well bypumping at high vacuum for 3 h.

Method A: To a stirring solution of the Boc protected aminoglycoside(0.054 mmol) in DCM (1 mL) were added 3 Å molecular sieves (4-6), andtrifluoroacetic acid (0.6 mL). The reaction was stirred at roomtemperature for 1 h, and checked for completeness by MS. Upon completionthe reaction mixture was diluted with ether (15 mL) to induceprecipitation. The vial was centrifuged and the supernatant wasdecanted. The precipitate was washed with ether (2×15 ml), decanted anddried under vacuum.

Method B: To a stirring solution of Boc-protected aminoglycosidederivative (0.078 mmol) in DCM (1.5 mL) at 0° C. was addedtrifluoroacetic acid (1.5 mL). The reaction was stirred for 45 minutes,and checked for completeness by MS. Upon completion, the reaction wasdiluted with dichloroethane (10 ml) and concentrated to dryness. Thelast dilution/concentration step was repeated twice.

Procedure 4: PyBOP Coupling

To a stirring solution of aminoglycoside derivative (0.078 mmol) in DMF(1 mL) at −40° C. was added the acid (0.16 mmol), followed by PyBOP(0.16 mmol) and DIPEA (0.31 mmol) and the reaction was stirred. Thereaction mixture was diluted with EtOAc (3 mL) and H₂O (3 mL), and theaqueous layer was separated and extracted with EtOAc (3×3 mL). Thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated to dryness.

Procedure 5: Epoxide Opening

To a stirring solution of the aminoglycoside derivative (0.06 mmol) inMeOH (2 mL) was added the epoxide (0.07 mmol), LiClO₄ (0.15 mmol), andthe reaction mixture was heated by microwave irradiation to 100° C. for90 minutes. The reaction progress was monitored by MS. Upon completion,the solvent was removed by rotary evaporation. The resulting residue wasdissolved in EtOAc (20 mL), washed with H₂O (2×5 mL) and brine (5 mL),dried over Na₂SO₄, filtered and concentrated to dryness.

Procedure 6: Phthalimido Deprotection

To a stirring solution of the phthalimido protected aminoglycoside(0.064 mmol) in EtOH (3 mL) was added hydrazine (0.32 mmol), and thereaction mixture was heated to reflux for 2 h. The reaction progress wasmonitored by MS. Upon cooling to room temperature, the cyclic by-productprecipitated and was removed by filtration. The filtrate wasconcentrated to dryness to yield a residue, which was dissolved in EtOAc(20 mL), washed with 5% NaHCO₃ (2×5 mL) and brine (5 mL), dried overNa₂SO₄, filtered and concentrated to dryness.

Procedure 7: Addition of Guanidinium GROUP

To a stirring solution of the aminoglycoside derivative (0.063 mmol) inDMF (1 mL) was added 1H-pyrazole-1-carboxamidine hydrochloride (0.09mmol), followed by DIPEA (0.862 ml) and the reaction mixture was heatedto 80° C. and stirred overnight. The reaction progress was monitored byMS. Upon completion, the reaction mixture was cooled to room temperatureand diluted with water (3 mL). The aqueous phase was separated andextracted with EtOAc (2×5 mL), and the combined organics were washedwith brine (5 mL), dried over Na₂SO₄, filtered and concentrated todryness.

Procedure 8: Sulfonylation

To a stirring solution of the aminoglycoside scaffold (0.067 mmol) inDCM (3 mL) was added DIPEA (0.128 mol) and the sulfonyl chloride (0.07mmol). The reaction mixture was stirred at room temperature and itsprogress was monitored by MS. Once complete, the solvent was removed byrotary evaporation and the residue was dissolved in ethyl acetate (20mL), washed with 5% NaHCO₃ (2×5 mL) and brine (5 mL), dried over Na₂SO₄,filtered and concentrated to dryness.

Procedure 9: N-Boc Protection

To a stirring solution of the amine (4.64 mmol) in THF (10 mL) was added1N NaOH (10 mL), followed by Boc-anhydride (5.57 mmol) and the reactionprogress was checked by MS. Once complete, the THF was removed by rotaryevaporation and water (40 mL) was added. The aqueous phase was separatedand extracted with Et₂O (2×30 ml). The aqueous phase was acidified to pH3 by the addition of dilute H₃PO₄ and was then extracted with EtOAc(2×60 ml). The combined organic layers were washed with H₂O (2×30 mL)and brine (30 mL), dried over Na₂SO₄, filtered and concentrated todryness.

Procedure 10: Syntheses of Epoxides

To a stirring solution of the alkene (5.16 mmol) in chloroform (20 mL)at 0° C. was added m-chloroperbenzoic acid (8.0 mmol) and the reactionmixture was stirred for 30 minutes at 0° C. and was then allowed to warmto room temperature. The reaction progress was monitored by MS and TLC,and additional portions of m-CPBA were added as needed. Upon completion,the reaction mixture was diluted with chloroform (50 mL) and washed with10% aq. Na₂SO₃ (2×30 mL), 10% aq. NaHCO₃ (2×50 mL) and brine (50 mL).The organic layer was dried over Na₂SO₄, filtered and concentrated toyield a crude product, which was purified by flash chromatography(silica gel/hexanes:ethyl acetate 0-25%).

Procedure 11: General Procedure for Synthesis of α-Hydroxy CarboxylicAcids

Step #1. O-(Trimethylsilyl) cyanohydrines: A 50-mL flask equipped with amagnetic stirring bar and drying tube was charged with the ketone oraldehyde (0.010 mmol), followed by THF (50 mL), trimethylsilyl cyanide(1.39 g, 14 mmol), and zinc iodide (0.090 g, 0.28 mmol), and thereaction mixture was stirred at room temperature for 24 hr. Solventevaporation gave a residue, which was dissolved in EtOAc (60 mL), washedwith 5% aq. NaHCO₃ (2×30 mL), H₂O (30 mL), and brine (30 mL), dried overNa₂SO₄, filtered and concentrated to dryness to yield a crude, which wascarried through to the next step without further purification.

Step #2. Acid hydrolysis to α-hydroxy carboxylic acid: AcOH (25 ml) andconc. HCl (25 ml) were added to the unpurified material from step #1 andthe reaction mixture was refluxed for 2-3 hr. The reaction mixture wasthen concentrated to dryness to give a white solid, which was carriedthrough to the next step without further purification.

Step #3. Boc protection: To a stirring solution of solid from step #2 in2 M NaOH (20 mL) and i-PrOH (20 mL) at 0° C. was added Boc₂O (6.6 g, 3mmol) in small portions, and the reaction mixture was allowed to warm toroom temperature over 4 h. i-PrOH was then evaporated, and H₂O (50 mL)was added, and the aqueous phase was separated and extracted with Et₂O(2×30 ml). The aqueous layer was acidified to pH 3 by addition of diluteH₃PO₄ and was extracted with EtOAc (2×60 ml). The combined organiclayers were washed with H₂O (2×30 mL) and brine (30 mL), dried overNa₂SO₄, filtered and concentrated to yield the desired N-Boc-α-hydroxycarboxylic acids in 56-72% yield.

Procedure 12: Protection of Amine by Fmoc Group

To a stirring solution of the amine (0.049 mol) in DCM (100 mL), wasadded DIPEA (16 mL, 0.099 mol) and the reaction mixture was cooled to 0°C. Fmoc-Cl (12.8 g, 0.049 mol) was then added portion-wise over severalminutes, and the reaction was allowed to warm to room temperature for 2hr. The organic layer was washed with water (2×50 mL) and brine (50 mL),dried over Na₂SO₄, filtered and concentrated to dryness to yield theFmoc protected amine (90-95% yield).

Procedure 13: Synthesis of Aldehydes via TEMPO/Bleach Oxidation

To a vigorously stirring solution of the alcohol (1.54 mmol) in DCM (4mL) was added TEMPO (0.007 g, 0.045 mmol, 0.03 mol %) and a 2M aqueousKBr solution (75 mL, 0.15 mmol, 10 mol %) and the reaction mixture wascooled to −10° C. In a separate flask NaHCO₃ (0.5 g, 9.5 mmol) wasdissolved in bleach (25 mL, Chlorox 6.0% NaOCl) to yield a 0.78 Mbuffered NaOCl solution. This freshly prepared 0.78 M NaOCl solution(2.3 mL, 1.8 mmol, 117 mol %) was added to the reaction mixture over 5min and the reaction was stirred for an additional 30 min at 0° C. Theorganic phase was separated and the aqueous layer was extracted withdichloromethane (2×4 mL). The combined organic layers were washed with10% aq. Na₂S₂O₃ (4 mL), sat. aq. NaHCO₃ (2×4 mL), brine (5 mL), driedover Na₂SO₄ and concentrated to dryness.

Procedure 14: Synthesis of Alcohols Via Borane Reduction

To a stirring solution of the acid (1.5 mmol) in THF (5 mL) at −10° C.was slowly added 1.0 M BH₃-THF (2.98 mL, 2.98 mmol). The reactionmixture was stirred vigorously for an additional 3 min at −10° C., andwas then allowed to warm to room temperature overnight. The reaction wasquenched by the dropwise addition of a solution of HOAc/H₂O (1:1 v/v,2.0 mL). The THF was removed by rotary evaporation and sat. aq. NaHCO₃(15 mL) was added. The aqueous layer was extracted with DCM (3×5 mL) andthe combined organic layers were washed with sat. aq. NaHCO₃ (2×5 mL),brine (10 mL), dried over Na₂SO₄, filtered and concentrated to dryness.

Procedure 15: Ozonolysis and Pinnick Oxidation

The substrate olefin (0.5 to 0.75 mmol) was dissolved in DCM (30 mL) andthe reaction was cooled to −78° C. Ozone was bubbled through until ablue color persisted (3 to 5 min), and the reaction was stirred for 1hr. Argon was then bubbled through to remove excess ozone for 10minutes. The reaction was further quenched by the addition of dimethylsulfide (10 equiv.), and was stirred for 30 min with warming to rt. Thesolvent was reduced under vacuum to yield the crude aldehyde, which wasdried under high-vacuum for 10 min, and used without furtherpurification. To a stirring solution of the aldehyde in THF, tBuOH andH₂O (3:3:2, 10 mL), was added NaH₂PO₄ (4 equiv.) followed by2-methyl-2-butene (10 equiv.) and sodium chlorite (2 equiv.), and thereaction was stirred for 4 hr. The reaction mixture was then added tosat. aq. NaCl (10 mL) and extracted with DCM (3×). The combined organiclayers were dried over Na₂SO₄, filtered and reduced under vacuum toyield a crude, which was purified by flash chromatography (silica gel.0→0.5 or 1% MeOH/DCM).

Procedure 16: Hydrogenolysis

To a stirring solution of aminoglycoside (0.031 mmol) in AcOH (2 mL),was added H₂O (1 mL), followed by Pd(OH)₂/C (40 mg) and the reaction wasstirred under a hydrogen atmosphere for 3 hours. The catalyst wasremoved by filtration, and the reaction was diluted with water andlyophilized to yield a crude, which was purified on a 1-inch reversephase HPLC column (buffered with 10 mM NH₄OH) to yield the desiredproduct.

General Purification Procedures

Method #1: Purification by Basic Condition

Mobile Phases:

A—Water with 10 mM NH₄OH

B—Acetonitrile with 10 mM NH₄OH

Columns:

A: Waters-XBridge Prep Shield RP18 Column

19×250 mm, 5 μm

Gradient: 20 min at 0%, then 0-20% in 200 min at a flow of 20 ml/min

B: Waters-XBridge Prep Shield RP18 Column

50×100 mm, 5 μm

Gradient: 20 min at 0%, then 0-20% in 200 min at a flow of 20 ml/min

Method #2: Purification by Acidic Condition

Mobile Phases:

A—Water with 0.1% TFA

B—Acetonitrile with 0.1% TFA

Columns:

A: Phenomenex Luna C18

21.4×250 mm, 10 μm

Gradient: 0-100%, flow 25 ml/min

B: Phenomenex Luna C18

50×250 mm, 10 μm

Gradient: 0-100%, flow 45 ml/min

Representative Intermediates

Aminoglycoside Freebasing

Amberlite IRA-400 (OH form) (200 g) was washed with MeOH (3×200 ml). Toa stirring suspension of the washed resin in MeOH (150 mL) was addedaminoglycoside sulfate (20 g) and the mixture was stirred overnight. Theresin was then filtered and washed with MeOH (100 mL) and the combinedorganic layers were concentrated to dryness to yield the desiredaminoglycoside.

(N-Hydroxy-5-norbornene-2,3-dicarboxyl-imido)-4-nitro-benzoate

To a stirring solution of 4-nitrobenzyl chloroformate (5.0 g, 0.023 mol)in THF (90 mL) at 0° C. was addedN-hydroxy-5-norbornene-2,3-dicarboximide (4.16 g, 0.023 mol), followedby the dropwise addition of a solution of Et₃N (3.2 mL, 0.02 mol) in THF(50 mL) and the reaction was stirred for 4 hours with gradual warming toroom temperature. The reaction vessel was then placed in the freezer(−5° C.) for 1 hour to induce precipitation of triethylaminehydrochloride, which was removed by filtration. The filtrate wasconcentrated to dryness to yield a residue, which was vigorously stirredin MeOH (80 mL) for 1 h and then filtered to yield(N-hydroxy-5-norbornene-2,3-dicarboxyl-imido)-4-nitro-benzoate as awhite solid (7.98 g, 0.022 mol, 96% yield): TLC (hexanes:EtOAc v/v 1:1)Rf=0.35.

2,5-Dioxo-pyrrolidin-1-yl-4-nitrobenzyl carbonate (PNZ-succinimide)

To a stirring solution of N-hydroxysuccinimide (5.35 g, 46.5 mmol) inanhydrous THF (100 mL) was added para-nitrobenzylchloroformate (10.0 g,46.5 mmol), and the solution was cooled in an ice bath. Triethylamine(6.5 mL, 4.89 g, 46.5 mmol) was added over 10 minutes, and, after 30minutes, the reaction mixture was allowed to warm to room temperatureand stir overnight. The slurry was cooled in an ice-bath, and wasfiltered, followed by rinsing with ethyl acetate. The filtrate wasconcentrated in vacuo, and the residue was triturated with methanol. Thesolids were isolated by filtration to give2,5-dioxopyrrolidin-1-yl-4-nitrobenzyl carbonate.

N,N′-bis-Cbz-2(S)-hydroxy-4-guanidino-butyric acid

To a stirring solution of 2(S)-hydroxy-4-amino-butyric acid (0.059 g,0.50 mmol) in DMF (2 ml) was addedN,N′-bis(benzyloxycarbonyl)-1H-pyrazole-1-carboxamidine (0.26 g, 0.70mmol) followed by DIPEA (0.87 mL, 4.99 mmol) and the reaction was heatedto 80° C. and stirred overnight. The crude mixture was purified on a2-inch reverse-phase HPLC column to yieldN,N′-bis-Cbz-2(S)-hydroxy-4-guanidino-butyric acid: MS: m/z (M+H)⁺calcd. 430.15. found 430.1.

Benzyl-2-(benzoyloxyamino)ethyl carbamate

To a solution of benzyl-N-(2-aminoethyl)carbamate chloride salt (1, 540mg, 2.34 mmol) in sat. aq. NaHCO₃ (45 mL) was added 1 M NaOH (15 mL) andthe reaction was stirred vigorously. DCM (30 mL) was added, followed bybenzoylperoxide (1.13 g, 4.68 mmol) and the reaction was stirredovernight. The organic layer was separated and washed with brine, driedover MgSO₄, filtered and concentrated to a crude, which was purified ona 1-inch reverse-phase HPLC column to yieldbenzyl-2-(benzoyloxyamino)ethyl carbamate (2, 252 mg, 0.80 mmol, 34.2%):MS: m/z (M+H)⁺ calc. 315.13, obs. 315.0.

Succinimidyl benzoyloxy(2-Cbz-aminoethyl)carbamate

To a stirring solution of disuccinimidyl carbonate (525 mg, 2.05 mmol)in CH₃CN (16 mL) was added benzyl-2-(benzoyloxyamino)ethyl carbamate (2,252 mg, 0.80 mmol) as a solution in CH₃CN (12 mL) over 4 hours, and thereaction was stirred overnight. Additional disuccinimidyl carbonate (251mg, 0.98 mmol) was added and the reaction was heated at 60° C.overnight. Solvent removal gave a crude, which was purified on a 2-inchreverse-phase HPLC column to yield succinimidylbenzoyloxy(2-Cbz-aminoethyl)carbamate (3, 81 mg, 0.18 mmol, 22.5%yield).

N-Boc-3-amino-2(5)-hydroxy-propionic acid

To a stirring solution of S-isoserine (4.0 g, 0.038 mol) in dioxane: H₂O(100 mL, 1:1 v/v) at 0° C. was added N-methylmorpholine (4.77 mL, 0.043mol), followed by Boc₂O (11.28 mL, 0.049 mol) and the reaction wasstirred overnight with gradual warming to room temperature. Glycine (1.0g, 0.013 mol) was then added and the reaction was stirred for 20 min.The reaction was cooled to 0° C. and sat aq. NaHCO₃ (75 mL) was added.The aqueous layer was washed with ethyl acetate (2×60 mL) and thenacidified to pH 1 with NaHSO₄. This solution was then extracted withethyl acetate (3×70 mL) and these combined organic layers were driedover Na₂SO₄, filtered and concentrated to dryness to give the desiredN-Boc-3-amino-2(S)-hydroxy-propanoic acid (6.30 g, 0.031 mmol, 81.5%yield): ¹H NMR (400 MHz, CDCl₃) δ 7.45 (bs, 1H), 5.28 (bs, 1H), 4.26 (m,1H), 3.40-3.62 (m, 2H), 2.09 (s, 1H), 1.42 (s, 9H); ¹³C NMR (100 MHz,CDCl3) δ 174.72, 158.17, 82, 71.85, 44.28, 28.45.

(N-Hydroxy-5-norbornene-2,3-dicarboxyl-imido)-tert-butyl-carbonate

To a stirring solution of N-hydroxy-5-norbornene-2,3-dicarboximide (20.0g, 0.112 mol) in THF (200 mL) at 0° C. was added triethylamine (0.65 mL,4.8 mmol), followed by the dropwise addition of a solution of Boc₂O(29.23 g, 0.134 mol) in THF (30 mL) and the reaction was allowed to stirovernight with gradual warming to room temperature. A precipitateformed, which was filtered and washed with cold THF (200 mL). The crudesolid was then vigorously stirred in MeOH (100 mL) for 1 hour, beforebeing filtered, washed with MeOH (50 mL), and dried under high vacuum toyield the desired(N-hydroxy-5-norbornene-2,3-dicarboxyl-imido)-tert-butylcarbonate as awhite solid (28.0 g, 0.1 mol, 89.3% yield): TLC (hexanes:ethyl acetate,1:1 v/v) R_(f)=0.44; NMR (400 MHz, DMSO-d₆) δ 6.10 (bs, 2H), 3.48 (bs,2H), 3.29-3.32 (m, 2H), 1.58-1.62 (m, 1H), 1.50-1.55 (m, 1H), 1.47 (s,9H).

N-Boc-4-amino-2(S)-hydroxy-butyric acid

To a stirring solution of S-4-amino-2-hydroxy-butyric acid (51.98 g,0.44 mol) in dioxane: H₂O (2 L, 1:1 v/v) was added K₂CO₃ (106 g, 0.91mol) followed by a solution of Boc-anhydride (100 g, 0.46 mol) indioxane (100 mL), and the reaction was stirred overnight. The reactionwas washed with DCM (2×300 mL), and the aqueous layer was acidified topH 2 with H₃PO₄. The aqueous layer was extracted with DCM (2×300 mL),and the combined organic layers were dried over MgSO₄, filtered andconcentrated to dryness to yield the desiredN-Boc-4-amino-2(S)-hydroxybutyric acid (48.2 g, 50% yield).

N-Boc-3-amino-propanal

To a stirring solution of 3-(Boc-amino)-1-propanol (25 mL, 0.144 mol) inwater saturated DCM (1.0 L) was added Dess-Martin reagent (99.2 g, 233.9mmol) and the reaction mixture was stirred for 1 hour. The reaction wasthen diluted with ether (1.0 L), followed by a solution of Na₂S₂O₃ (250g) in 80% NaHCO₃ (450 g in 1.0 L H₂O). The reaction was stirredvigorously for 30 minutes until two layers formed, the top layer wasclear. The reaction was filtered to remove the precipitated solids andthe aqueous layer was extracted with ether (1.0 L). The organic layerwas washed with sat. NaHCO₃ (1.0 L), H₂O (1.0 L), and brine (1 L), driedover Na₂SO₄ and concentrated to a clear oil. The crude oil was dissolvedin EtOAc:hexanes (1:1 v/v, 1.0 L) and filtered through a short silicagel column to yield the desired N-Boc-3-amino-propanal (21.7 g, 0.125mol, 85.6% yield): ¹H NMR (400 MHz, CDCl₃) δ 9.77 (s, 1H, CHO), 4.85(bs, 1H, NH), 3.36-3.42 (m, 2H, CH₂), 2.67 (t, 2H, CH₂), 1.39 (s, 9H,(CH₃)₃).

N-Boc-1-oxa-6-azaspiro[2.5]octane

4-Methylene-piperidine (0.222 g, 1.12 mmol) was submitted to Procedure10 to form the desired N-Boc-1-oxa-6-azaspiro[2.5]octane (0.215 g, 1.01mmol, 90.2% yield): ¹H NMR (250 MHz, DMSO-d₆) δ 3.29-3.61 (m, 6H),1.56-1.70 (m, 2H), 1.30-1.54 (m, 11H).

2-(Pent-4-enyl)-isoindoline-1,3-dione

To a stirring solution of 5-bromo-pentene (6.0 g, 0.040 mol) in DMF (30mL) was added K₂CO₃ (4.7 g, 0.034 mol) and potassium phthalimide (6.21g, 0.033 mmol) and the reaction mixture was heated at 100° C. for 1 hr.The reaction mixture was cooled to room temperature, and water (50 mL)was added. The aqueous layer was then extracted with ethyl acetate (2×50mL), and the combined organic layers were washed with 5% aq. NaHCO₃(2×20 mL), brine (30 mL) and dried over Na₂SO₄. Filtration and solventevaporation gave an oil, which was purified by flash chromatography(silica gel/hexanes:ethyl acetate 0-35%) to yield the desired2-(pent-4-enyl)-isoindoline-1,3-dione as a solid (6.36 g, 0.029 mmol,72.5% yield): MS m/e [M+H]⁺ calcd 216.1. found 216.1; NMR (250 MHz,DMSO-d₆) δ 7.79-7.95 (m, 4H), 5.70-5.91 (m, 1H), 4.90-5.11 (m, 2H), 3.58(t, 2H), 1.98-2.10 (m, 2H), 1.59-1.78 (m, 2H).

2-(3-(Oxiran-2-yl)-propyl)-isoindoline-1,3-dione

2-(Pent-4-enyl)-isoindoline-1,3-dione (6.36 g, 0.029 mmol) was submittedto Procedure 10 for epoxide formation to yield2-(3-(oxiran-2-yl)-propyl-isoindoline-1,3-dione (5.8 g, 0.025 mmol,86.2% yield): MS m/e [M+H]⁺ calcd 232.1. found 232.1; ¹H NMR (250 MHz,DMSO-d₆) δ 7.75-7.90 (m, 4H, Ar), 3.52 (t, 2H, CH₂), 2.87-2.96 (m, 1H,CH), 2.70 (t, 1H), 2.30-2.45 (m, 1H), 1.36-1.80 (m, 4H).

N-Boc-3-hydroxypyrrolidine-3-carboxylic acid

N-Boc-3-pyrrolidone (0.010 mmol) was submitted to Procedure 11 to yieldthe desired N-Boc-3-hydroxy-pyrrolidine-3-carboxylic acid.

N-Boc-1-amino-but-3-ene

3-Buten-1-amine (4.93 g, 0.069 mol) was submitted to Procedure 9 for Bocprotection to yield a crude, which was purified by flash chromatography(silica gel/hexanes:ethyl acetate 0-30%) to yieldN-Boc-1-amino-but-3-ene (6.47 g, 0.038 mol, 55.1% yield).

N-Boc-2-(oxiran-2-yl)-ethyl carbamate

N-Boc-1-amino-but-3-ene (6.47 g, 0.038 mol) was submitted to Procedure10 for epoxide formation to yield a crude, which was purified by flashchromatography (silica gel/hexanes:ethyl acetate 0-45%) to yieldN-Boc-2-(oxiran-2-yl)-ethyl carbamate (6.0 g, 0.032 mol, 84.2% yield):¹H NMR (250 MHz, DMSO-d₆) δ 2.98-3.09 (m, 2H), 2.83-2.92 (m, 1H), 2.65(t, 1H), 2.42 (dd, 1H), 1.44-1.66 (m, 2H), 1.36 (s, 9H, (CH₃)₃).

N-Boc-3-hydroxy-azetidin-3-carboxylic acid

N-Boc-3-azetidinone (21.9 g, 0.128 mol) was submitted to Procedure 11 toyield the desired N-Boc-3-hydroxy-azetidin-3-carboxylic acid (18.7 g,0.086 mol, 67.0% yield): MS m/e [M+H]⁺ calcd 218.1. found 218.2.

3-Methylene-1-methylamino-cyclobutane

To a stirring solution of 3-methylene-1-cyano-cyclobutane (2.5 g, 0.026mol) in THF (35 ml) at 0° C. was slowly added 2M LiAlH₄ (22 mL, 0.044mmol) and the reaction was allowed to warm to room temperature. Thereaction was then quenched by the addition of sat. aq. NH₄Cl (10 mL),and THF (10 mL). The organic layer was separated and concentrated todryness to yield a residue, which was dissolved in ethyl acetate (100mL). The organic layer was washed with 5% NaHCO₃ (2×20 mL), brine (20mL), dried over Na₂SO₄, filtered and concentrated to yield the desired3-methylene-1-methylamino-cyclobutane as an oil, which was carriedthrough to the next step without further purification.

3-Methylene-1-N-Boc-methylamino-cyclobutane

To a stirring solution of 3-methylene-1-methylamino-cyclobutane (2.52 g,0.026 mol) in 1N NaOH (15 ml) and THF (15 mL), was added Boc₂O (6.7 g,0.030 mol) and the reaction mixture was stirred overnight. THF wasevaporated and the aqueous layer was extracted with ethyl acetate (2×40mL). The combined organic layers were washed with 5% NaHCO₃ (2×20 mL)brine (20 mL), dried over Na₂SO₄, filtered and concentrated to drynessto yield a crude, which was purified by flash chromatography (silicagel/hexanes:ethyl acetate 0%-60%) to yield the desired3-methylene-1-N-Boc-methylamino-cyclobutane (1.9 g, 0.0096 mol, 36.9%yield): ¹H NMR (250 MHz, DMSO-d₆) δ 6.88 (bs, 1H), 4.72 (s, 2H),2.95-3.05 (m, 2H), 2.56-2.71 (m, 2H), 2.21-2.40 (m, 3H), 1.20 (s, 9H).

N-Boc-1-oxaspiro[2.3]hexan-5-yl-methanamine

3-Methylene-1-N-Boc-methylamino-cyclobutane (1.9 g, 0.0096 mol) wassubmitted to Procedure 10 for epoxide formation to yieldN-Boc-1-oxaspiro[2.3]hexan-5-yl-methanamine (1.34 g, 6.27 mol, 65.3%yield): ¹H NMR (250 MHz, DMSO-d₆) δ 2.99-3.10 (m, 2H), 2.60-2.66 (m 2H),1.99-2.47 (m, 5H), 1.40 (s, 9H).

N-Fmoc-4-amino-butyraldehyde diethyl acetal

4-Amino-butyraldehyde diethyl acetal (8.0 g, 0.050 mol) was Fmocprotected following Procedure 12 to give the desiredN-Fmoc-4-amino-butyraldehyde diethyl acetal (22.08 g, MS m/e [M+Na]⁺calcd 406.2. found 406.1), which was carried through to the next stepwithout further purification.

N-Fmoc-4-amino-butyraldehyde

To a stirring solution of N-Fmoc-4-amino-butyraldehyde diethyl acetal(0.050 mmol) in 1,4-dioxane (100 mL) was added aq. HCl (100 ml, 1:1 v/v,H₂O: conc. HCl) and the reaction progress was monitored by MS. Uponcompletion, the organic solvent was removed by rotary evaporation, andthe aqueous layer was extracted with ethyl acetate (2×200 mL). Thecombined organic layers were washed with 5% NaHCO₃ (2×75 mL), brine (75mL), dried over Na₂SO₄, filtered and concentrated to dryness to yieldthe desired N-Fmoc-4-amino-butyraldehyde (15.35 g, 0.049 mol, 90.0%yield), which was carried through to the next step without furtherpurification: MS m/e [M+Na]⁺ calcd 332.1. found 332.0.

3-Methylene-cyclobutane carboxylic acid

To a stirring solution of KOH (70.0 g, 1.25 mol) in EtOH/H₂O (500 mL,1:1 v/v) was added 3-methylenecyclobutane carbonitrile (25.0 g, 026 mol)and the reaction mixture was refluxed for 6 h. The reaction progress wasmonitored by TLC and, upon completion, the mixture was cooled andacidified to pH 3-4 with HCl. The ethanol was evaporated, and theremaining aqueous layer was extracted with Et₂O (200 mL). The organiclayer was washed with water (2×20 mL), brine (30 ml), dried over Na₂SO₄,filtered and concentrated to dryness to yield 3-methylene-cyclobutanecarboxylic acid, which was carried through to the next step withoutfurther purification: ¹H NMR (250 MHz, CDCl₃) δ 10.75 (bs, 1H), 4.80 (s,2H), 2.85-3.26 (m, 5H).

N-Boc-3-Methylene-cyclobutanamine

To a stirring solution of 3-methylene-cyclobutane carboxylic acid (1.0g, 8.9 mmol) in THF (90 mL) was added NaN₃ (2.0 g, 31.1 mmol), followedby tetrabutyl ammonium bromide (0.48 g, 1.5 mmol) and Zn(OTf)₂ (0.1 g,0.3 mmol), and the reaction mixture was heated to 40° C. Boc₂O (2.1 g,9.8 mmol) was then added at once, and the reaction was heated at 45° C.overnight. The reaction was then cooled to 0° C. and was quenched with10% aq. NaNO₂ (180 mL). The THF was evaporated and the aqueous layer wasextracted with EtOAc (180 mL). The organic layer was washed with 5% aq.NaHCO₃ (2×20 mL), brine (30 ml), dried over Na₂SO₄, filtered andconcentrated to dryness to yield a crude, which was purified by flashchromatography (silica gel/hexanes:ethyl acetate: 0-90%) to yield thedesired N-Boc-3-methylene-cyclobutanamine (0.57 g, 3.1 mmol, 34.9%yield): ¹H NMR (250 MHz, CDCl3) δ 4.83 (s, 2H), 4.79 (bs, 1H), 4.05-4.23(m, 1H), 2.92-3.11 (m, 2H), 2.50-2.65 (m, 2H), 1.44 (s, 9H).

N-Boc-1-oxaspiro[2.3]hexan-5-amine

N-Boc-3-methylene-cyclobutanamine (1.65 g, 9.0 mmol) was submitted toProcedure 10 for epoxide formation to yieldN-Boc-1-oxaspiro[2.3]hexan-5-amine (1.46 g, 7.33 mmol, 81.5% yield): ¹HNMR (250 MHz, CDCl₃) δ 4.79 (bs, 1H), 4.13-4.31 (m, 1H), 2.66-2.83 (m,4H), 2.31-2.47 (m, 2H), 1.45 (s, 9H).

N-Boc-2,2-dimethyl-3-amino-propionaldehyde

N-Boc-3-amino-2,2-dimethyl propanol (0.415 g, 2.04 mmol) was submittedto Procedure 13 to yield N-Boc-2,2-dimethyl-3-amino-propionaldehyde(0.39 g. 1.94 mmol, 95.1% yield): ¹H NMR (250 MHz, CDCl₃) δ 9.42 (s,1H), 4.80 (bs, 1H). 3.11 (d, 2H), 1.39 (s, 9H), 1.06 (s, 6H).

N-Boc-3-amino-3-cyclopropyl propionaldehyde

N-Boc-3-amino-propanol (0.130 g, 0.60 mmol) was submitted to Procedure13 for oxidation to the corresponding N-Boc-3-amino-3-cyclopropylpropionaldehyde, which was carried through to the next step withoutfurther purification.

4(S)-tert-Butyldimethylsilyloxy-N-Boc-pyrrolidin-2(R)-carboxaldehyde

4(S)-tert-Butyldimethylsilyloxy-N-Boc-pyrrolidin-2(R)-methanol (0.50 g,1.50 mmol) was submitted to Procedure 13 for oxidation to thecorresponding4(S)-tert-butyldimethylsilyloxy-N-Boc-pyrrolidin-2(R)-carboxaldehyde,which was carried through to the next step without further purification.

3-tert-Butyldimethylsilyloxy-propanal

3-tert-Butyldimethylsilyloxy-propanol (0.50 g, 2.62 mmol) was submittedto Procedure 13 for oxidation to the corresponding3-tert-butyldimethylsilyloxy-propanal, which was carried through to thenext step without further purification.

2-Methyl-N-Boc-2-amino-propanal

2-Methyl-N-Boc-2-amino-propanol (0.83 g, 4.38 mmol) was submitted toProcedure 13 for oxidation to the corresponding2-methyl-N-Boc-2-amino-propanal (0.706 g, 3.77 mmol, 86.1% yield): ¹HNMR (250 MHz, CDCl₃) δ 9.40 (s, 1H), 1.57 (s, 1H), 1.41 (s, 9H), 1.30(s, 6H).

N-Boc-1-amino-cyclobutane carboxylic acid

1-Amino-cyclobutane carboxylic acid ethyl ester (1.0 g, 6.28 mmol) wasdissolved in 1N HCl (10 mL) and the reaction was heated to a reflux for2 hours. The reaction mixture was then concentrated to dryness to yielda crude which was submitted to Procedure 9 for Boc protection to yieldthe desired N-Boc-1-Amino-cyclobutane carboxylic acid.

N-Boc-1-amino-cyclobutyl-methanol

N-Boc-1-amino-cyclobutane carboxylic acid (6.28 mmol) was submitted toProcedure 14 for reduction to the correspondingN-Boc-1-Amino-cyclobutyl-methanol.

N-Boc-1-amino-cyclobutane carboxaldehyde

N-Boc-1-amino-cyclobutyl-methanol (0.25 g, 1.24 mmol) was submitted toProcedure 13 to yield the corresponding N-Boc-1-amino-cyclobutanecarboxaldehyde (0.24 g, 1.20 mmol, 96.8% yield): ¹H NMR (250 MHz, CDCl3)δ 9.0 (s, 1H), 4.91 (bs, 1H), 3.74 (bs, 2H), 1.71-2.20 (m, 4H), 1.42 (s,9H).

N-Boc-3-amino-cyclobutanone

To a vigorously stirring solution of N-Boc-3-methylene-cyclobutanamine(9.8 g, 53.5 mmol) in DCM (160 mL) and H₂O (160 mL) was added K₂CO₃ (3g, 21.7 mmol), followed by NaIO₄ (35 g, 163.5 mmol), tetrabutylammoniumchloride (0.2 g, 0.72 mmol) and RuCl₃ (0.6 g, 7.6 mmol). During thecourse of the reaction, the organic solution turned dark brown, thecatalyst turned black, while the upper aqueous layer turned white. Thereaction was monitored by TLC, and upon completion, the reaction mixturewas filtered through a pad of celite. The filtrates were transferred toa separatory funnel, and the aqueous layer was extracted with DCM (2×50mL). The combined organic layers were washed with 5% NaHCO₃ (2×30 mL),brine (30 mL), dried over Na₂SO₄, filtered and evaporated to dryness toyield a crude, which was purified by flash chromatography (silicagel/hexanes:ethyl acetate 0-60%) to yield the desiredN-Boc-3-amino-cyclobutanone (7.13 g, 38.53 mmol, 72% yield): NMR (250MHz, CDCl₃) δ 4.88 (bs, 1H), 4.13-4.29 (m, 1H), 3.23-3.41 (m, 2H),2.9-3.05 (m, 2H), 1.39 (s, 9H).

N-Boc-1-hydroxy-3-amino-cyclobutyl-carboxylic acid

N-Boc-3-amino-cyclobutanone (7.13 g, 38.53 mmol) was submitted toProcedure 11 to yield the desiredN-Boc-1-hydroxy-3-amino-cyclobutyl-carboxylic acid (MS m/e [M+H]⁺ calcd232.1. found 232.2.

N,N-diBoc-4(S)-amino-2(S)-methanol-pyrrolidine

N,N-diBoc-4(S)-amino-pyrrolidine-2(S)-carboxylic acid (1.03 g, 3.12mmol) was submitted to Procedure 14 to yield the correspondingN,N-diBoc-4(S)-amino-2(S)-methanol pyrrolidine (0.605 g, 1.91 mmol,61.2% yield), which was carried through to the next step without furtherpurification.

N,N-diBoc-4(S)-amino-pyrrolidine-2(5)-carbaldehyde

N,N-diBoc-4(S)-amino-2(S)-methanol pyrrolidine (0.486 g, 1.53 mmol) wassubmitted to Procedure 13 for oxidation to the correspondingN,N-diBoc-4(S)-amino-pyrrolidine-2(S)-carbaldehyde, which was carriedthrough to the next step without further purification.

N-Boc-1-aminomethyl-cyclopropyl-methanol

N-Boc-1-aminomethyl-cyclopropane carboxylic acid (1.0 g, 4.64 mmol) wassubmitted to Procedure 14 to yield the correspondingN-Boc-1-aminomethyl-cyclopropyl-methanol (0.99 g, MS m/e [M+H]⁺ calcd202.1. found 202.1), which was carried through to the next step withoutfurther purification.

N-Boc-1-aminomethyl-cyclopropane carboxaldehyde

N-Boc-1-aminomethyl-cyclopropyl-methanol (0.87 g, 4.32 mmol) wassubmitted to Procedure 13 for oxidation to the correspondingN-Boc-1-aminomethyl-cyclopropane carboxaldehyde, which was carriedthrough to the next step without further purification.

N-Boc-1-amino-cyclopropyl-methanol

N-Boc-1-amino-cyclopropane carboxylic acid (0.25 g, 1.24 mmol) wassubmitted to Procedure 14 to yield the correspondingN-Boc-1-amino-cyclopropyl-methanol (0.051 g, 0.27 mmol, 21.8% yield),which was carried through to the next step without further purification.

N-Boc-1-amino-cyclopropane carboxaldehyde

N-Boc-1-amino-cyclopropyl-methanol (0.051 g, 0.27 mmol) was submitted toProcedure 13 for oxidation to the correspondingN-Boc-1-amino-cyclopropane carboxaldehyde, which was carried through tothe next step without further purification.

N-Boc-1(R)-amino-2(S)-tert-butyldimethylsilyloxy-cyclopentane-4(S)-carboxylicacid

To a stirring solution ofN-Boc-1(R)-amino-2(S)-hydroxy-cyclopentane-4(S)-carboxylic acid methylester (0.622 g, 2.40 mmol) in DCM (1.9 mL) was added imidazole (0.164 g,2.41 mmol), DMAP (0.047 g, 0.35 mmol) and TBSCl (0.363 g, 2.40 mmol) andthe reaction was stirred at room temperature for 18 hours, followed byheating at 40° C. for 1 hour. The reaction mixture was cooled to roomtemperature, and was quenched with H₂O (3 mL). The organic layer wasseparated and was concentrated to dryness to yield a residue, which wasdissolved in isopropanol (6 mL) and 1M NaOH (2.9 mL), and the reactionwas heated at 60° C. for 1 hour. The reaction was cooled to 0° C. andslowly acidified to pH 3 with 1M HCl (3 mL). After adding chloroform (18mL), the organic layer was separated, dried over Na₂SO₄, andconcentrated to dryness to yield the desired acid (0.75 g, 2.09 mmol,87.1% yield).

N-Boc-1(R)-amino-2(S)-tert-butyldimethylsilyloxy-4(S)-hydroxymethyl-cyclopentane

N-Boc-1(R)-amino-2(S)-tert-butyldimethylsilyloxy-cyclopentane-4(S)-carboxylicacid (0.53 g, 1.47 mmol) was submitted to Procedure 14 for reduction tothe correspondingN-Boc-1(R)-amino-2(S)-tert-butyldimethylsilyloxy-4(S)-hydroxymethyl-cyclopentane(0.44 g, 1.27 mmol, 86.4% yield): ¹H NMR (250 MHz, CDCl₃) δ 4.69-4.79(m, 1H), 4.08-4.13 (m, 1H), 3.88 (bs, 1H), 3.52-3.61 (m, 2H), 2.16-2.30(m, 2H), 1.96-2.14 (m, 2H), 1.48-1.53 (m, 2H), 1.47 (s, 9H), 0.91 (s,9H), 0.09 (s, 6H).

N-Boc-1(R)-amino-2(S)-tert-butyldimethylsilyloxy-cyclopentane-4(S)-carboxaldehyde

N-Boc-1(R)-amino-2(S)-tert-butyldimethylsilyloxy-4(S)-hydroxymethyl-cyclopentane(0.44 g, 1.27 mmol) was submitted to Procedure 13 for oxidation to thecorrespondingN-Boc-1(R)-amino-2(S)-tert-butyldimethylsilyloxy-cyclopentane-4(S)-carboxaldehyde(0.42 g, 1.22 mmol, 96.1% yield).

tert-Butyl-2-(N-Boc-3-hydroxy-azetidin-3-yl)acetate

To a stirring solution of N-Boc-3-azetidinone (0.45 g, 2.64 mmol) in THF(5 mL) was slowly added a 0.5 M solution of2-tert-butoxy-2-oxoethyl-zinc chloride in Et₂O (10 mL, 5.0 mmol), andthe reaction mixture was stirred for 5 h. The reaction was then quenchedwith sat. aq. NH₄Cl (10 mL), and the aqueous layer was separated andextracted with ethyl acetate (2×30 mL). The combined organic layers werewashed with 5% aq. NaHCO₃ (2×10 mL), brine (15 mL), dried over Na₂SO₄,filtered and concentrated to dryness to yieldtert-butyl-2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetate (MS m/e [M+H]⁺calcd 288.2. found 287.7).

2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetic acid

To a stirring solution oftert-butyl-2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetate (0.86 g, 2.99 mmol)in dioxane (18 mL) was added 3M HCl (5 mL), and the mixture was heatedat 70° C. for 1 h. The reaction mixture was then cooled to 0° C. and itwas basified with 2 M NaOH (8 mL), followed by addition of BOC₂O (1.0 g,4.6 mmol). The reaction mixture was allowed to warm to room temperaturefor 2 h, and was then concentrated to half its total volume on therotary evaporator. Isopropanol (3 mL) and chloroform (12 mL) were thenadded and the mixture was cooled to 0° C. and slowly acidified to pH 3with 1M HCl. The organic layer was then separated, dried over Na₂SO₄,and concentrated to dryness to yield2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetic acid (0.65 g, 2.81 mmol, 94.0%yield).

N-Boc-3-(2-hydroxy-ethyl)-azetidin-3-ol

2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetic acid (0.44 g, 1.90 mmol) wassubmitted to Procedure 14 for reduction to yield the correspondingN-Boc-3-(2-hydroxy-ethyl)-azetidin-3-ol (0.29 g, 1.33 mmol, 70.0%yield).

2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetaldehyde

N-Boc-3-(2-hydroxy-ethyl)-azetidin-3-ol (0.29 g, 1.33 mmol) wassubmitted to Procedure 13 for oxidation to the corresponding2-(N-Boc-3-hydroxy-azetidin-3-yl)-acetaldehyde, which was carriedthrough to the next step without further purification.

N-Boc-3-hydroxymethyl-azetidine

N-Boc-azetidine-3-carboxylic acid (1.94 g, 9.64 mmol) was submitted toProcedure 14 for reduction to the correspondingN-Boc-3-hydroxymethyl-azetidine, which was carried through to the nextstep without further purification.

N-Boc-azetidine-3-carboxaldehyde

N-Boc-3-hydroxymethyl-azetidine (9.64 mmol) was submitted to Procedure13 for oxidation to the desired N-Boc-azetidine-3-carboxaldehyde, whichwas carried through to the next step without further purification.

2-(N-Boc-azetidin-3-yl)-2-hydroxy-acetic acid

N-Boc-azetidine-3-carboxaldehyde (1.60 g, 8.64 mmol) was submitted toProcedure 11 to yield the desired2-(N-Boc-azetidin-3-yl)-2-hydroxy-acetic acid (MS m/e [M+H]⁺ calcd232.1. found 231.8).

Synthesis of (2R,3R)-4-azido-2-benzyloxy-3-fluorobutanoic acid (5)

Molecular sieves (4 Å, 4 g) were added to a round bottom flask, and wereactivated by heating with a Bunsen burner under high vacuum. DCM (100mL) was then added and the flask was cooled to −35° C. with acryocooler. Titanium tetraisopropoxide (1.75 mL, 5.95 mmol) and(R,R)-(−)-diisopropyl tartrate (1.65 mL, 7.75 mmol) were added and thereaction was stirred for 30 min. Penta-1,4-dienol (5 g, 59.4 mmol) andexcess cumene hydroperoxide (80%, 17.5 mL) were added in small portions,and stirring was continued at −35° C. for 48 hr. The reaction wasquenched by addition of sat. aq. Na₂SO₄ (5 mL) immediately followed byEt₂O (50 mL) and the reaction was stirred for 2 hr with warming to rt.The reaction mixture was filtered through Celite, and washed with Et₂O.Solvent removal under vacuum without heating resulted in approximately30 mL of a yellow solution. Excess cumene alcohol and hydroperoxide wereremoved by flash chromatography (silica gel, 40% Et₂O/hex). Finallysolvent removal under vacuum without heating yielded a mixture of(2S,3R)-1,2-epoxy-4-penten-3-ol (1) (Rf=0.47, 1:1 EtOAc/hex) anddiisopropyl tartrate (Rf=0.6), which was used in the next step withoutfurther purification.

To a stirring solution of epoxide (1) in THF (100 mL) under an argonatmosphere was added tetrabutylammonium iodide (2.2 g, 5.96 mmol),followed by benzyl bromide (8.6 mL, 71.9 mmol) and the reaction wascooled to −15° C. Sodium hydride (60% in mineral oil, 2.65 g, 66.1 mmol)was added in small portions and the reaction was stirred overnight withwarming to rt. The reaction was quenched with MeOH, filtered throughCelite, and washed with Et₂O. Solvent removal gave an oily residue whichwas purified by flash chromatography (silica gel, 5→10% Et₂O/hex) toyield (2S,3R)-1,2-epoxy-3-benzyloxy-4-pentene (2) as a clearnon-volatile liquid (5.3 g, 47.6% yield): Rf=0.69 (1:4 EtOAc/hex);=−36.7° (c 1.52, CHCl₃); HRMS (ESI) (M+H)⁺ calc. for C₁₂H₁₄O₂ 191.1067,obs. 191.1064; ¹H NMR (CDCl₃, 300 MHz) δ 7.38-7.33 (m, 5H), 5.92-5.78(m, 1H), 5.41-5.39 (m, 1H), 5.37-5.33 (m, 1H), 4.66 (d, J=11.95 Hz, 1H),4.49 (d, J=11.96 Hz, 1H), 3.83 (dd, J=7.34, 4.20 Hz, 1H), 3.10 (dt,J=4.07, 4.06, 2.70 Hz, 1H), 2.79 (dd, J=5.21, 4.00 Hz, 1H), 2.70 (dd,J=5.23, 2.64 Hz, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ 138.32, 134.67, 128.56(2C), 127.87 (2C), 127.82, 119.73, 79.54, 70.83, 53.41, 45.00.

NaN₃ (3.38 g, 52 mmol) and NH₄Cl (2.78 g, 52 mmol) in H₂O (10 mL) wereheated until a clear solution was obtained. This solution was then addeddropwise to a solution of (2S,3R)-1,2-epoxy-3-benzyloxy-4-pentene (2)(3.3 g, 17.4 mmol) in MeOH (200 mL) and the reaction mixture was stirredfor 4 days. The organic solvent was removed under vacuum, and theaqueous layer was extracted with DCM (3×). The combined organic layerswere dried over Na₂SO₄, filtered and reduced under vacuum to yield acrude, which was purified by flash chromatography (silica gel, 10→20%Et₂O/hex) to yield (2S,3R)-1-azido-3-benzyloxy-4-penten-2-ol (3) (2.66g, 66% yield) as a non-volatile clear liquid: Rf=4.8 (1:4 EtOAc/hex);HRMS (EST) (M+Na)⁺ calc. for C₁₂H₁₅N₃O₂ 256.1056, obs. 256.1057; =-46.3°(c 1.50, CHCl₃); ¹H NMR (CDCl₃, 300 MHz) δ 7.42-7.28 (m, 5H), 5.91-5.76(m, 1H), 5.46 (dd, J=17.16, 1.42 Hz, 1H), 5.42 (dd, J=24.00, 1.37 Hz,1H), 4.65 (d, J=11.67 Hz, 1H), 4.39 (d, J=11.67 Hz, 1H), 3.88-3.80 (m,2H), 3.44-3.40 (m, 2H), 2.22 (d, J=3.60 Hz, 1H); ¹³C NMR (CDCl₃, 100MHz) δ 137.88, 134.60, 128.66 (2C), 128.08 (2C), 128.05, 121.40, 81.39,72.61, 70.70, 53.0; FTIR (NaCl): 3435, 2870, 2102, 1642, 1454, 1070cm⁻¹.

To a stirring solution of DAST (900 μL, 6.87 mmol) in benzene (3.2 mL)and pyridine (400 μL) in a plastic container at −10° C. was added(2S,3R)-1-azido-3-benzyloxy-4-penten-2-ol (3) (750 mg, 3.21 mmol) insmall portions, and the reaction was stirred at this temperature for 48hr followed by 6 hr at rt. The reaction mixture was slowly added to sat.aq. NaHCO₃ (20 mL) at 0° C. and was stirred for 10 min. The resultingaqueous mixture was extracted with DCM (3×) and the combined organiclayers were washed with 2 N HCl, dried over MgSO₄, filtered and reducedunder vacuum to yield a crude, which was purified by flashchromatography (silica gel, 1% Et₂O/hex) to yield(3R,4R)-5-azido-4-fluoro-3-benzyloxy-pent-1-ene (4) (128 mg, 16.9%yield) as a nonvolatile clear liquid: Rf=0.63 (1:9 EtOAC/Hex);[α]_(D)=−11.9° (c 1.50, CHCl₃); ¹H NMR (CDCl₃, 400 MHz)

7.44-7.29 (m, 5H), 4.63 (dddd, J=47.64, 7.07, 4.99, 3.32 Hz, 1H),5.49-5.42 (m, 2H), 4.70 (d, J=11.95 Hz, 1H), 4.57 (ddd, J=7.07, 4.99,3.32 Hz, 1H), 4.44 (d, J=11.90 Hz, 1H), 4.03 (ddd, J=16.87, 7.57, 5.04Hz, 1H), 3.64-3.52 (m, 1H), 3.45 (ddd, J=27.45, 13.63, 3.27 Hz, 1H). ¹⁹FNMR (CDCl₃, 282 MHz) −196.66 (dddd, J=47.27, 27.08, 19.84, 16.89 Hz);¹³C NMR (CDCl₃, 100 MHz) δ 137.80, 133.09 (d, J=5.30 Hz), 128.70 (2C),128.09 (3C), 121.04, 93.33 (d, J=181.54 Hz), 79.08 (d, J=20.39 Hz),70.92, 51.46 (d, J=22.25 Hz). FTIR (NaCl): 2930, 2104, 1643, 1454, 1281,1115, 1069 cm⁻¹.

(3R,4R)-5-azido-4-fluoro-3-benzyloxy-pent-1-ene (4) (128 mg, 0.543 mmol)was submitted to Procedure 15, followed by recrystallization from hothexanes (2×) to yield (2R,3R)-4-azido-2-benzyloxy-3-fluorobutanoic acid(5) (120 mg, 90%): [α]_(D)=−56.9° (c 0.68, CHCl₃); HRMS (ESI negativemode) (M−H) calc. for C₁₁H₁₂FN₃O₃ 252.0790, obs. 252.0782; ¹H NMR(CDCl₃, 400 MHz)

10.55 (s, 1H), 7.46-7.34 (m, 5H), 4.98 (dddd, J=46.40, 7.57, 4.91, 2.92Hz, 1H), 4.94 (d, J=11.47 Hz, 1H), 4.55 (d, J=11.51 Hz, 1H), 4.17 (dd,J=27.26, 2.86 Hz, 1H), 3.77 (dt, J=13.89, 13.66, 7.27 Hz, 1H), 3.42(ddd, J=24.28, 13.20, 4.92 Hz, 1H); ¹⁹F NMR (CDCl₃, 376 MHz)

−198.36 (dddd, J=46.28, 27.22, 24.46, 14.15 Hz); ¹³C NMR (CDCl₃, 100MHz) δ 174.63 (d, J=4.21 Hz), 136.37, 129.15 (2C), 129.07, 128.98 (2C),91.53 (d, J=182.59 Hz), 76.40 (d, J=19.90 Hz), 73.96 (s), 50.87 (d,J=25.13 Hz); FTIR (NaCl): 3151, 2098, 1753, 1407, 1283, 1112 cm⁻¹.

Synthesis of ent-5

Starting from penta-1,4-dienol (5 g, 59.4 mmol) and using(S,S)-(+)-diisopropyl tartrate under the same reaction conditions asdescribed above the enantiomer ent-2 was obtained (4.9 g, 43% yield):[α]_(D)=+35.7° (c 1.76, CHCl₃). (2R,3S)-1,2-Epoxy-3-benzyloxy-4-pentene(ent-2, 3.9 g, 20.5 mmol) was submitted to the same reaction conditionsdescribed above to yield the enantiomer(2R,3S)-1-azido-3-benzyloxy-4-penten-2-ol (ent-3, 2.75 g, 57% yield):MD=+47.3° (c 1.30, CHCl₃). (2R,3S)-1-Azido-3-benzyloxy-4-penten-2-ol(ent-3) (500 mg, 2.14 mmol) was submitted to the same reactions asdescribed above to yield the enantiomer(3S,4S)-5-azido-4-fluoro-3-benzyloxy-pent-1-ene (ent-4, 75.5 mg, 0.32mmol, 15% yield, [α]_(D)=+10.7°, c 1.50, CHCl₃), which was submitted tothe same reaction conditions as described above to yield ent-5 (59 mg,73% yield): [α]_(D)=+58.6° (c 0.73, CHCl₃).

Synthesis of (R)-4-Azido-3,3-difluoro-2-benzyloxy-butanoic acid (3)

To a stirring solution of DMSO (690 μL, 9.65 mmol) in DCM (25 mL) at−78° C. was added oxalyl chloride (3.21 mL of a 2.0 M solution in DCM,6.43 mmol) and the reaction was stirred for 1 hr. A solution of(2S,3R)-1-azido-3-benzyloxy-4-penten-2-ol (1) (750 mg, 3.21 mmol) in DCM(1 mL) was added dropwise and the reaction mixture was stirred for 1 hrat −78° C. N-Methyl morpholine (1.41 mL, 12.9 mmol) was added dropwise,and the reaction was stirred at −15° C. for 2 hr. The reaction wasquenched with phosphate buffer (0.1 M, pH 6.0) and the aqueous layer wasseparated. The organic layer was washed with the phosphate buffer (3×),dried over Na₂SO₄, filtered and reduced under vacuum to give a brownresidue. The residue was dissolved in Et₂O, dried over MgSO₄, filteredthrough a cotton plug, and reduced under vacuum to yield the crudeketone, which was dissolved in DCM (1 mL) and was added to a stirringsolution of DAST (2 mL, 15.3 mmol) in DCM (3 mL) in a plastic vial at−25° C. The reaction was allowed to slowly warm to rt and was stirredfor 48 hr. The reaction mixture was then slowly poured into stirringsat. aq. NaHCO₃ (20 mL) at 0° C., and was stirred for 10 min. Theresulting aqueous mixture was extracted with DCM (3×), and the combinedorganic layers were dried over Na₂SO₄, filtered and reduced under vacuumto yield a crude, which was purified by flash chromatography (silicagel, 1% Et₂O/hex) followed by preparative TLC purification (silica gel,0.5 mm, 5% Et₂O/hex) to yield(R)-5-azido-4,4-difluoro-3-benzyloxy-pent-1-ene (2, 193 mg, 0.76 mmol,24% yield), as a non-volatile clear liquid: Rf=0.72 (1:4 EtOAc/hex);[α]_(D)=−23.8° (c 1.52, CHCl₃); ¹H NMR (CDCl₃, 300 MHz)

7.44-7.31 (m, 5H), 5.89 (dddd, J=16.88, 10.61, 7.11, 0.62 Hz, 1H),5.59-5.56 (m, 1H), 5.53 (d, J=10.74 Hz, 1H), 4.71 (d, J=11.67 Hz, 1H),4.50 (d, J=11.66 Hz, 1H), 4.14 (td, J=14.25, 7.13, 7.13 Hz, 1H), 3.64(tq, J=13.67, 13.67, 13.67, 11.19, 11.19 Hz, 2H); ¹⁹F NMR (CDCl₃, 282MHz) δ −116.63 (dtd, J=257.62, 13.91, 13.90, 8.72 Hz), −111.27 (dtd,J=257.59, 16.18, 16.16, 7.04 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ 137.14,130.33 (t, J=3.06, 3.06 Hz), 128.71 (2C), 128.27, 128.20 (2C). 122.78,120.69 (dd, J=249.89, 246.83 Hz), 78.87 (dd, J=30.35, 25.35 Hz), 71.48(d, J=0.48 Hz), 51.47 (dd, J=30.26, 25.92 Hz); FTIR (NaCl): 2928, 2108,1455, 1292, 1091 cm⁻¹.

(R)-5-Azido-4,4-difluoro-3-benzyloxy-pent-1-ene (2, 193 mg, 0.76 mmol)was submitted to Procedure 15, followed by washing with cold hexanes(3×) at −20° C. to yield (3) (139 mg, 67.6% yield): [α]_(D)=−32.4° (c0.80, CHCl₃); FIRMS (ESI negative mode) (M−H) for C₁₁H₁₁F₂N₃O₃ 270.0696,obs. 270.06924; ¹H NMR (CDCl₃, 400 MHz)

7.46-7.32 (m, 5H), 6.48 (s, 1H). 4.84 (d, J=11.30 Hz, 1H), 4.67 (d,J=11.30 Hz, 1H), 4.37 (dd, J=12.23, 9.78 Hz, 1H), 3.75 (dd, J=14.67,12.35 Hz, 2H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −112.61 (qd, J=260.95, 12.30,12.29, 12.29 Hz), −109.68 (dtd, J=260.79, 14.75, 14.68, 9.94 Hz); ¹³CNMR (CDCl₃, 100 MHz) δ 170.84, 135.48, 129.01, 128.94 (2C), 128.78 (2C),119.59 (t, J=251.58, 251.58 Hz), 76.56 (dd, J=29.86, 27.24 Hz), 74.34,51.58 (dd, J=28.94, 26.76 Hz). FTIR (NaCl): 3337, 2929, 2112, 1738,1455, 1292, 1210, 1119 cm⁻¹.

Synthesis of ent-3

(2R,3S)-1-Azido-3-benzyloxy-4-penten-2-ol (ent-1, 500 mg, 2.14 mmol) wassubmitted to the same reaction conditions described above to yield(S)-5-azido-4,4-difluoro-3-benzyloxy-pent-1-ene (ent-2, 114 mg, 21%yield, [α]_(D)=+27.9° (c 3.14, CHCl₃)). Ent-2 (75.5 mg, 0.32 mmol) wassubmitted to Procedure 15 to yield(S)-4-azido-2-benzyloxy-3,3-difluorobutanoic acid (ent-3, 34.8 mg, 43%yield, [α]_(D)=+36.4° (c 0.80, CHCl₃).

Synthesis of (2S,3S)-4-azido-2,3-bis-benzyloxybutanoic acid (3)

To a stirring solution of (2S,3R)-1-azido-3-benzyloxy-4-penten-2-ol (1)(250 μL, 1.07 mmol) in THF (50 mL) under argon was addedtetrabutylammonium iodide (42 mg, 0.11 mmol) followed by benzyl bromide(155 μL, 1.27 mmol) and the reaction was cooled to 0° C. Sodium hydride(60% in mineral oil, 47 mg, 1.18 mmol) was added in small portions andthe reaction was stirred overnight with warming to rt. The reaction wasquenched with MeOH, filtered through Celite, and washed with Et₂O. Theorganic solvent was removed under vacuum to give an oily residue, whichwas purified by flash chromatography (silica gel, 2% Et₂O/hex) to yield(3R,4S)-5-azido-3,4-bisbenzyloxy-pent-1-ene (2, 237 mg, 65% yield) as aclear non-volatile liquid: Rf=−0.62 (1:4 EtOAc/hex); [α]_(D)=−6.1° (c1.50, CHCl₃); ¹H NMR (CDCl₃, 300 MHz) δ 7.35-7.24 (m, 10H), 5.81 (ddd,J=17.15, 10.58, 7.45 Hz, 1H), 5.37 (ddd, J=5.70, 1.65, 0.86 Hz, 1H),5.33 (ddd, J=12.07, 1.44, 0.81 Hz, 1H), 4.63 (s, 2H), 4.61 (d, J=11.87Hz, 1H), 4.35 (d, J=11.78 Hz, 1H), 3.90 (tdd, J=7.37, 5.65, 0.79, 0.79Hz, 1H), 3.60 (ddd, J=6.39, 5.69, 3.64 Hz, 1H), 3.43 (dd, J=12.93, 6.42Hz, 1H), 3.35 (dd, J=12.93, 3.60 Hz, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ138.25, 138.01, 135.43, 128.60 (4C), 128.29 (2C), 128.02, 127.99 (2C),127.87, 119.97, 80.76, 80.23, 73.33, 70.79, 51.69; FTIR (NaCl): 2867,2100, 1606, 1454, 1286, 1095, 1073.

(3R,4S)-5-azido-3,4-bis-benzyloxy-pent-1-ene (2, 237 mg, 0.69 mmol) wassubmitted to Procedure 15 to yield(2S,3S)-4-azido-2,3-bis-benzyloxybutanoic acid (3, 187.7 mg, 75% yield):[α]_(D)=−15.1° (c 1.05, CHCl₃); HRMS (ESI negative mode) (M−H) calc. forC₁₈H₁₉N₃O₄ 340.1303, obs. 340.1296; ¹H NMR (CDCl₃, 300 MHz)

7.24 (s, 1H), 7.38-7.33 (m, 10H), 4.79 (d. J=11.61 Hz, 1H), 4.66 (s,2H), 4.56 (d, J=11.61 Hz, 1H), 4.20 (d, J=4.24 Hz, 1H), 3.98 (td,J=6.56, 4.30, 4.30 Hz, 1H), 3.58 (dd, J=13.04, 6.62 Hz. 1H), 3.42 (dd,J=13.04, 4.31 Hz, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 175.57, 137.92, 137.34,129.44 (2C), 129.36 (2C), 129.15, 129.04 (2C), 128.98 (2C), 128.94,79.71, 77.651, 74.04, 73.89, 51.65; FTIR (NaCl): 3000, 2918, 2103, 1722,1455, 1284, 1110 cm⁻¹.

Synthesis of ent-3

(2R,3S)-1-azido-3-benzyloxy-4-penten-2-ol (ent-1, 250 mg, 1.07 mmol) wassubmitted to the same reaction conditions as described above to yield(3S,4R)-5-azido-3,4-bis-benzyloxy-pent-1-ene (ent-2, 322 mg, 59% yield):[α]_(D)=+7.9° (c 1.50, CHCl₃). Ent-2 (178 mg, 0.55 mmol) was submittedto Procedure 15 to yield ent-3 (144 mg, 77% yield): [α]_(D)=+15.2° (c0.81, CHCl₃).

Synthesis of Compound 9

Synthesis of Epoxy Alcohol Ent-2

A 3-neck, 5 liter round bottomed flask equipped with an overheadmechanical stirrer, a thermocouple probe and a nitrogen inlet/outlet wascharged with powdered, freshly activated molecular sieves (4 Å, 84 g,0.8 wt. equiv), followed by anhydrous dichloromethane (2.1 L, 20 vol).The resulting suspension was cooled to approximately −42° C. using anacetonitrile/CO₂ bath, then titanium tetraisopropoxide (37 mL, 0.125mol, 10 mol %) was charged into the batch, followed by(S,S)-(+)-diisopropyl tartrate (35 mL, 0.166 mol. 13.3 mol %). Thereaction mixture was stirred for 30 minutes, then divinyl alcohol 1 (105g, 1.25 mol, 1.0 equiv) was added over 3 minutes using an additionfunnel (minor exotherm, 2° C.). Cumene hydroperoxide (370 mL, 80% titer,1.99 mol, 1.59 equiv) was then added to the batch over 5 minutes usingan addition funnel (10° C. exotherm). The reaction was allowed toproceed for 18 hours, holding the temperature between −45 and −30° C.When complete as determined by TLC analysis (R_(f) 0.42 for divinylalcohol, and 0.18 for epoxy alcohol, 50% MTBE in Heptanes), the reactionwas quenched with saturated aqueous sodium sulfate (105 mL, 1 vol),diluted with MTBE (1.05 L, 10 vol) and the batch allowed to warm toambient temperature, with vigorous stirring. Diatomaceous earth, Celite®(105 g, 1 wt. equiv) was added to the batch, which was then filteredthrough a pad of Celite®. The filter cake was washed with MTBE (0.5 L)and the filtrate concentrated in vacuo on a rotary evaporator (withwater bath held at 10-20° C.) to afford a yellow/brownish oil. A portionof the crude product [311 g] was subjected to silica plug (1 kg silicagel) using 0-60% MTBE/petroleum ether. The fractions containing theproduct were collected and concentrated to obtain a colorless oil (48.3g). This material was then purified via column chromatography (300 gsilica gel, 5-30% MTBE/petroleum ether) to afford ent-2 as a clearliquid [22.6 g, 36% overall mass recovery]: R_(f)=0.59 (1:1MTBE/petroleum ether); ¹H NMR (CDCl₃, 500 MHz) δ 5.85 (ddd, J=17.0,10.5, 6.2 Hz, 1H), 5.40 (dt, J=17.3, 1.3 Hz, 1H), 5.27 (dt, J=10.5, 1.3Hz, 1H), 4.36-4.33 (m, 1H), 3.10 (ddd, J=3.8, 3.8, 3.0 Hz, 1H), 2.81(dd, J=2.9, 5.0 Hz, 1H), 2.76 (dd, 4.1, 5.0 Hz, 1H), 2.07 (d, J=3.0 Hz,1H).

Synthesis of Compound 3

The reaction was carried out at 20-g scale of alcohol following aliterature procedure J. Org. Chem. 2009, 74(15), 5758-5761). A 2-Lround-bottomed flask equipped with a mechanical stirrer, a thermocoupleprobe, and an addition funnel was charged with a solution of epoxyalcohol ent-2 [20 g, 200 mmol, 1 equiv] in tetrahydrofuran (400 mL, 20vol) along with Ph₃P (105 g, 400 mmol, 2 equiv), and 4-nitrobenzoic acid(67 g, 400 mmol, 2 equiv) under a nitrogen atmosphere. DIAD (81 g, 400mmol, 2 equiv) was added to the reaction mixture using an additionfunnel while maintaining the reaction mixture at 0° C. (ice bath). Oncethe addition of DIAD was complete, the cold bath was removed and thereaction mixture was allowed to come to ambient temperature (23° C.).The reaction mixture was stirred for 1.5 h (all starting materialconsumed) and then quenched with aqueous NaHCO₃ solution (100 ml, 5 vol)followed by the addition of MTBE (1000 mL, 50 vol). The resultingsolution was transferred into a separatory funnel. Brine (100 mL, 5 vol)was added to obtain phase separation. The organic phase was washed withbrine (2×20 vol), dried (MgSO₄), and concentrated under vacuum to obtainan oil (296 g). The oil was passed through a silica plug (1 kg) using10-20% MTBE/heptanes. The crude solid (46 g) was then dissolved intoMTBE (20 vol) and washed with NaHCO₃ (3×5 vol), water (2×2 vol), brine(2×2 vol), dried (MgSO₄), concentrated, and further dried to obtain thebenzoate ester as a white solid [29 g, 59%: R_(f)=0.56 (1:1MTBE/heptanes)]; ¹H NMR (CDCl₃, 500 MHz) δ 8.35 (d, J=10.8 Hz, 2H), 8.25(d, J=10.8 Hz, 2H), 5.97 (ddd, J=17.2, 10.6, 6.2 Hz, 1H), 5.48 (td,J=17.3, 1.2 Hz, 1H), 5.40 (td, J=10.7, 1.1 Hz, 1H), 5.34 (dd, J=5.0, 1.3Hz, 1H), 3.31 (ddd, J=6.5, 4.1, 2.6 Hz, 1H), 2.93 (dd, J=4.2, 4.2 Hz,1H), 2.76 (dd, J=4.8, 2.6 Hz, 1H).

The hydrolysis of the benzoate ester was carried out following theliterature procedure (J. Org. Chem. 2009, 74(15), 5758-5761). Thussolution of the ester (22.7 g, 91 mmol, 1 equiv) in methanol (340 mL, 15vol) was treated with an aqueous solution of K₂CO₃ (13.8 g, 100 mmol,1.1 equiv, in 34 mL, 1.5 vol water) at 10-15° C. The solutionimmediately turned into a thick slurry. The slurry was stirred atambient temperature (23° C.) for 3 h (starting material consumed). Thereaction mixture was concentrated on a rotary evaporator (at ambientwater bath temperature) to ˜2 vol (45 mL). The thick solution was thenreslurried in DCM (454 mL, 20 vol). The slurry was filtered and thesolids were washed with DCM (2×5 vol, 2×114 mL). The combined organicfiltrate was dried (MgSO₄), filtered, and concentrated to obtain a solid(31 g). The crude material was then purified by column chromatography(silica gel, 10-30% MTBE/petroleum ether) to obtain the desired alcohol3 as a clear oil [9.24 g, quantitative yield, R_(f)=0.31 (1:1MTBE/heptanes)]; ¹H NMR (CDCl₃, 300 MHz) δ 5.94 (ddd, J=16.2, 10.6, 5.5,1H), 5.40 (d, J=17.3 Hz, 1H), 5.26 (d, J=10.6 Hz, 1H), 4.0 (t, J=5.3 Hz,1H), 3.07 (m, 1H), 2.84 (t, J=4.8 Hz, 1H), 2.77-2.74 (m, 1H), 2.57 (brs, 1H).

Synthesis of Compound 4

A 1-L three-necked round-bottomed flask equipped with an additionfunnel, an overhead mechanical stirrer, a nitrogen inlet/outlet, wascharged with alcohol 3 [9.24 g, 92.3 mmol, 1 equiv] in anhydroustetrahydrofuran (166 mL, 18 vol). The solution was cooled to −10 to −15°C. The catalyst Bu₄NI (3.41 g, 9.23 mmol, 10 mol %) was charged into thereactor followed by benzyl bromide (19.1 g, 112 mmol, 1.2 equiv). Theresulting solution was stirred for 20 min. Sodium hydride (4.1 g, 1.1equiv, 60% mineral oil dispersion) was then added to the batch inportions such that the batch temperature was maintained at −10 to −15°C. Once the addition of sodium hydride was complete, the reactionmixture was stirred for an additional 30 min and then the cold bath wasremoved and reaction mixture brought up to ambient temperature andfurther stirred for 18 h. The reaction was quenched with aqueous NaHCO₃(37 mL, 4 vol) while maintaining the temperature at −5 to 0° C. (icebath). The resulting solution was diluted with MTBE (185 mL, 20 vol),the organic layer was washed with water (2×18 mL, 2×3 vol), brine (1×18mL, 1×3 vol), dried (MgSO₄), filtered, and concentrated under reducedpressure to obtain crude product as an oil. The synthesis was repeatedon 1.98 g scale of alcohol 3. The crude from both the reactions werecombined and purified via column chromatography (silica gel column,2.5-10% MTBE/heptanes) to obtain the desired benzylated product 4 as anoil [13.96 g, 65%: R_(f)=9 0.61 (3:7 MTBE/heptanes)]; ¹H NMR (CDCl₃, 500MHz) δ 7.36-7.32 (m, 4H), 7.29-7.26 (m, 1H), 5.83 (ddd, J=17.3, 10.5,6.7, 1H), 5.36 (td, J=17.3, 1.4 Hz, 1H), 5.31 (td, J=10.5, 1.2 Hz, 1H),4.63 (ABq, J=12.0 Hz, 2H). 3.62 (ddd, J=, 1H), 3.11-3.08 (m, 1H), 2.78(t, J=4.4 Hz, 1H), 2.60 (dd. J=5.0, 2.7 Hz, 1H).

Synthesis of Compound 5

A 250-mL round-bottomed flask equipped with a reflux condenser wascharged with alcohol 4 [10 g, 52.5 mmol, 1 equiv], phthalimide (11.6 g,78.8 mmol, 1.5 equiv), pyridine (0.85 mL, 10.5 mmol, 20 mol %) and IPA(100 mL, 10 vol) and the resulting solution was stirred at 80-82° C. for8 hrs. The reaction mixture was then cooled to ambient temperature andconcentrated on a rotatory evaporator to dryness. The residue wasadsorbed on silica gel (20 g), dried under high vacuum and then purifiedby flash column chromatography on silica gel (10-40% MTBE/heptanes) toafford the desired phthalimide protected amino alcohol 5 as a whitetacky solid [15.85 g, 89%]: R_(f)=0.34 (1:1 MTBE/heptanes); ¹H NMR(DMSO-d₆, 500 MHz) δ 7.84-7.82 (m, 4H), 7.36-7.31 (m, 4H), 7.28-7.25 (m,1H), 5.93 (ddd, J=17.5, 10.5, 10.1 Hz, 1H). 5.38-5.35 (m, 2H), 5.12 (d,J=5.5 Hz, 1H), 4.53 (d, J=11.9 Hz, 1H), 4.40 (d, J=11.9 Hz, 1H), 3.98(dddd, J=9.0, 4.5, 4.5, 4.5 Hz 1H), 3.86 (dd, J=5.8, 4.6 Hz, 1H), 3.67(dd, J=13.7, 8.9 Hz, 1H), 3.59 (dd, J=13.7, 4.4 Hz, 1H).

Synthesis of Compound 6

A 1-L three-necked round-bottomed flask equipped with an additionfunnel, an overhead mechanical stirrer, and a nitrogen inlet/outlet wascharged with a solution of alcohol 5 [15 g, 44.5 mmol, 1 equiv] inanhydrous tetrahydrofuran (270 mL, 18 vol). The solution was cooled to−10 to −15° C., then Bu₄NI (1.64 g. 4.45 mmol, 10 mol %) was chargedinto the reactor followed by benzyl bromide (9.2 g, 53.8 mmol, 1.2equiv). The resulting solution was stirred for 20 min, then sodiumhydride (1.97 g, 1.1 equiv, 60% mineral oil dispersion) was added to thebatch in portions such that the batch temperature was maintained at −10to −15° C. Once the addition of sodium hydride was complete, thereaction mixture was stirred for an additional 30 min and then broughtto ambient temperature and further stirred for 18 h. The reaction wasquenched with aqueous NaHCO₃ (60 mL, 4 vol) while maintaining thereaction mixture at −5 to 0° C. (ice bath). The reaction mixture wasthen diluted with MTBE (300 mL, 20 vol) and the phases separated. Theorganic layer was washed with water (2×45 mL, 2×3 vol), brine (1×45 mL,1×3 vol), dried (MgSO₄), filtered, and concentrated to obtain the crudeproduct as an oil. The synthesis was repeated on 1.75 g scale of alcohol5. The combined crude products from both reactions were purified byflash column chromatography on silica gel (5-25% MTBE/heptanes) toobtain the desired product 6 as a semi solid [15.1 g, 71%: R_(f)=0.61(1:1 MTBE/heptanes)]; ¹H NMR (CDCl₃, 300 MHz) δ 7.74-7.71 (m, 2H),7.67-7.64 (m, 2H), 7.37-7.27 (m, 5H), 7.10-7.07 (m, 2H), 6.98-6.93 (m,3H), 5.97 (ddd, J=17.5, 10.4, 10.0 Hz, 1H), 5.42 (d, J=4.38 Hz, 1H),5.38 (s, 1H), 4.68 (dd, J=12.3, 12.3 Hz, 2H), 4.45 (d, J=5.37 Hz, 1H),4.41 (d, J=5.58 Hz, 1H), 3.99-3.82 (m, 3H), 3.65 (dd, J=13.6, 3.2 Hz,1H).

Synthesis of Aldehyde 7 and Carboxylic Acid 8

A solution of alkene, 6 [1 g, 2.34 mol] in DCM (60 mL, 60 vol) wassparged with ozone at <−70° C. (dry ice-acetone) for 25 min using houseair as oxygen source to generate the ozone. Once the reaction was deemedcompete (TLC, 1:1 MTBE/heptanes), the solution was sparged with nitrogenfor 20 min to remove residual ozone. The reaction was quenched withdimethyl sulfide (1.7 mL, 23.4 mmol, 10 equiv) while maintaining thereaction mixture at <−70° C. (dry ice-acetone). The cold bath wasremoved and the mixture was allowed to warm to ambient temperature. Thereaction mixture was concentrated under reduced pressure and furtherdried under high vacuum to obtain the crude aldehyde as a thick oil(1.12 g, >99%, R_(f)=0.36, 1:1 MTBE/heptanes). The reaction was repeatedat 13 g scale of 6. The two lots of crude aldehyde were combined andsubjected to the Pinnick oxidation without further purification.

The crude aldehyde 7 [14.06 g], was taken into a mixture oftetrahydrofuran, tBuOH, and water (105 mL, 105 mL, 70 mL, 3:3:2, 20 vol)along with NaH₂PO₄ (15.6 g, 130 mmol, 4 equiv) and 2-methyl-2-butene(34.4 mL, 324 mmol, 10 equiv). The solution was cooled (15±5° C., waterbath). Sodium chlorite (3.9 g, 43 mmol, 1.33 equiv) was added to thebatch and the resulting solution was stirred at ambient temperature for4 hr. The completion of the reaction was confirmed by TLC analysis (1:1MTBE/heptanes and 5% MeOH in DCM). The reaction was then quenched withbrine (280 mL, 20 vol) and the product extracted into DCM (3×280 mL,3×20 vol). The organic layers were dried (MgSO₄), concentrated underreduced pressure to obtain the crude acid as a thick oil. The crude acidwas purified by flash column chromatography over silica (5-100%MTBE/heptanes followed by 5-20% MeOH/DCM). Fractions containing the acidwere combined and concentrated under reduced pressure to afford acid 8as a white solid [2.64 g, 18%: R_(f)=0.33, 5:95 MeOH/DCM)]; NMR (CDCl₃,500 MHz) δ 7.78 (dd, J=5.5, 3.0 Hz, 2H), 7.70 (dd, J=5.5, 3.0 Hz, 2H),7.43-7.40 (m, 2H), 7.37-7.29 (m, 3H), 7.20-7.19 (m, 2H), 7.14-7.11 (m,2H), 7.09-7.05 (m, 1H), 4.76 (d, J=11 Hz, 1H), 4.65 (dd, J=10.9, 9.4 Hz,2H), 4.55 (d, J=11.8 Hz, 1H), 4.13 (ddd, J=6.2, 6.2, 3.1 Hz, 1H), 4.1(d, J=3.0 Hz, 1H), 3.98 (dd, J=14.2, 6.2 Hz, 1H), 3.89 (dd, J=14.2, 6.2Hz, 1H).

Synthesis of Compound 9

A round bottomed flask equipped with a magnetic stirring bar, and athermocouple probe was charged with a solution of phthalimide-protectedamino acid 8 [2.5 g, 5.61 mmol, 1.0 equiv] in THF (28 mL, 11 vol, bulksolvent grade). To the clear, yellow solution was added deionized water(15 mL, 6 vol) and the resulting mixture cooled to 5° C. Methylaminesolution in water (5.0 mL, 40 wt %, 56.1 mmol, 10 equiv) was then addedto the batch, which was warmed to ambient temperature (21-23° C.) andstirred for 22.5 hours. Analysis of an aliquot from the reaction mixtureby LCMS indicated the reaction was complete. The reaction mixture wasthen concentrated in vacuo to a yellow solid residue, removing allexcess methylamine. The residue was taken up in THF (60 mL, 24 vol) andwater (30 mL, 12 vol), cooled to 0-5° C., and to the crude amino acidsolution was added potassium carbonate (3.9 g, 28.26 mmol, 5.0 equiv),followed by benzylchloroformate (1.4 mL, 9.81 mmol, 1.75 equiv). Thebatch was warmed to ambient temperature and the reaction allowed toproceed for 25.5 hours. Analysis of an aliquot at this time point byLCMS indicated a complete conversion of the amino acid to the carbamate.The reaction mixture was concentrated under reduced pressure to removemost of THF, the aqueous residue was diluted with water (30 mL, 12 vol)and the pH adjusted with 2N HCl to approximately pH 5 (pH paper strip).The crude product was extracted with chloroform (3×60 mL), the extractswashed with water (1×60 mL) and with aqueous NaCl (1×60 mL), dried(MgSO₄) and concentrated in vacuo to a yellow, mobile oil (3.52 g) whichwas purified by flash column chromatography on silica gel (50 wt. equiv;elution with 0-5% MeOH in CHCl₃) to afford 9 as a yellow oil, whichpartially solidified upon further drying under high vacuum [2.22 g,88.1% yield over two steps]. ¹H NMR (DMSO, 500 MHz) δ 12.92 (s, 1H),7.43-7.23 (m, 15H), 5.04 (s, 2H), 4.67 (d, J=11.10 Hz, 1H), 4.58 (d,J=11.10 Hz, 1H), 4.48 (d, J=11.05 Hz, 1H), 4.42 (d, J=11.05 Hz, 1H),4.09 (d, J=2.95 Hz, 1H), 3.96 (ddd, J=6.30, 6.30, 3.15 Hz, 1H), 3.29(dd, J=6.30, 6.30, 2H).

Synthesis of Cyclopropyl Amino Acids

Ethyl-2-(tert-Butyldimethylsilyloxy)acrylate (2)

A solution of ester 1 (4.00 g, 34.4 mmol) and triethylamine (4.79 mL,34.4 mmol) in anhydrous dichloromethane (170 mL) was cooled to 0° C.under nitrogen and tert-butyldimethylsilyltrifluoromethane sulfonate(8.31 mL, 36.2 mmol) was added dropwise. The resulting solution wasstirred vigorously at reflux for 4 h. The solvent was then carefullyevaporated, the residue was dissolved in Et₂O (170 mL), and the organicphase was washed with water (3×50 mL). The organic phase was dried(Na₂SO₄), filtered, and concentrated. The residue was purified by silicagel chromatography eluting with 0-20% diethyl ether/hexanes to afford 2(4.89 g, 62%) as a clear oil: ¹H NMR (500 MHz, CDCl₃) δ 5.50 (d, J=1.0Hz, 1H), 4.85 (d, J=1.0 Hz, 1H), 4.21 (q, J=7.0 Hz, 2H), 1.31 (t, J=7.0Hz, 3H), 0.95 (s, 9H), 0.16 (s, 6H).

2-tert-Butyl-1-Ethyl-1-(tert-butyldimethylsilyloxy)cyclopropane-1,2-dicarboxylate(3a and 3b)

A mixture of ethyl-2-(tert-butyldimethylsilyloxy)acrylate (2, 500 mg,2.17 mmol) and Cu(acac)₂ (0.011 g, 0.043 mmol) was heated at 80° C. Asolution of tert-butyl diazoacetate (463 mg, 3.25 mmol) in benzene (5mL) was added to the reaction mixture over 2 h. After this time, thereaction mixture was cooled to room temperature and concentrated. Theresidue was purified by silica gel chromatography eluting with 0-10%diethyl ether/hexanes to afford both diastereomers 3a (0.119 g, 16%) and3b (0.235 g, 31%) as clear oils. 3a: ¹H NMR (500 MHz, CDCl₃) δ 4.25-4.13(m. 2H), 2.28 (dd, J=7.5, 2.0 Hz, 1H), 1.73 (dd, J=7.5, 2.0 Hz, 1H),1.59 (dd, J=9.5, 4.0 Hz, 1H). 1.46 (s, 9H), 1.29 (t, J=7.5 Hz, 3H), 0.90(s, 9H), 0.18 (s, 3H), 0.12 (s, 3H); ESI MS m/z 367 [M+Na]; 3b: ¹H NMR(500 MHz, CDCl₃) δ 4.23 (dq, J=11.0, 7.0 Hz, 1H), 4.13 (dq, J=11.0, 7.0Hz, 1H), 2.11 (dd, J=10.0, 1.5 Hz, 1H), 1.85 (dd, J=5.5, 2.5 Hz, 1H),1.43 (s, 9H), 1.54 (dd, J=10.0, 4.0 Hz, 1H), 1.28 (t, J=7.5 Hz, 3H),0.86 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H); ESI MS m/z 367 [M+Na]⁺.

2-(tert-Butyldimethylsilyloxy)-2-(ethoxycarbonyl)cyclopropanecarboxylicAcid (4a and 4b)

A mixture of dicarboxylate 3a and 3b (0.385 g, 1.12 mmol, 1:2 ratio of3a/3b), trifluoroacetic acid (0.43 mL), and dichloromethane (0.5 mL) wasstirred overnight at room temperature. The solids were filtered, and thefiltrate was concentrated. The residue was purified by silica gelchromatography eluting with 0-100% diethyl ether/hexanes to afford bothdiastereomers 4a (0.050 g, 15%) and 4b (0.078 g, 24%) as off-whitesolids. 4a: ¹H NMR (500 MHz, CDCl₃) δ 4.25-4.17 (m, 2H), 2.38 (dd,J=7.5, 1.5 Hz, 1H), 1.81-1.76 (m, 2H), 1.30 (t, J=7.0 Hz, 3H), 0.90 (s,9H), 0.21 (s, 3H), 0.13 (s, 3H); ESI MS m/z 289 [M+H]⁺; 4b: ¹H NMR (500MHz, CDCl₃) δ 4.22 (q, J=7.0 Hz, 1H), 2.21 (dd, J=10.0, 1.5 Hz, 1H),1.93 (dd, J=8.0, 2.0 Hz, 1H), 1.52 (dd, J=6.0, 3.5 Hz. 1H), 1.28 (t,J=7.0 Hz, 3H), 0.87 (s, 9H), 0.19 (s, 3H), 0.17 (s, 3H); ESI MS m/z 287[M−H]⁻.

Ethyl-2-(Benzyloxycarbonylamino)-1-(tert-butyldimethylsilyloxy)cyclopropanecarboxylate(5b)

A mixture of2-(tert-butyldimethylsilyloxy)-2-(ethoxycarbonyl)cyclopropanecarboxylicacid (4b, 0.335 g, 1.16 mmol) in toluene (5 mL) under nitrogen wastreated with Hüenig's base (0.260 mL, 1.51 mmol) and the mixture wascooled to 0° C. After this time, DPPA (0.324 mL, 1.51 mmol) was addedand the mixture was heated at 90° C. for 30 min, followed by theaddition of benzyl alcohol (0.155 mL. 1.51 mmol). After 15 h, themixture was cooled, diluted with ethyl acetate (75 mL), and washedsequentially with 10% citric acid (2×50 mL), water (50 mL), andsaturated NaHCO₃ (50 mL). The organic phase was dried (MgSO₄), filtered,and concentrated. The residue was purified by silica gel chromatographyeluting with 10% EtOAc/hexanes to 100% EtOAc to afford the titlecompound as a clear oil (0.146 g, 30%): ¹H NMR (300 MHz, CDCl₃) δ7.34-7.30 (m, 5H), 5.40-5.38 (m, 1H), 5.21-5.00 (m, 2H), 4.29-4.18 (m,2H), 4.16-4.09 (m, 1H), 1.50-1.47 (m, 2H), 1.30 (t, J=7.2 Hz, 3H), 0.88(s, 9H), 0.26-0.07 (m, 6H); Multimode (APCI+ESI) MS m/z 295 [M+H]⁺.

Ethyl 2-(Benzyloxycarbonylamino)-1-hydroxycyclopropanecarboxylate (6b)

To a solution of ethyl2-(benzyloxycarbonylamino)-1-(tert-butyldimethylsilyloxy)cyclopropanecarboxylate(1.45 g, 3.69 mmol) in THF (35 mL) under N₂ was added HF.pyridine (1.0mL, 38 mmol). The reaction mixture was stirred for 5 h. After this time,additional HF.pyridine (1.0 mL, 38 mmol) was added and stirring wascontinued for 19 h. The reaction mixture was then cooled to 0° C. anddiluted with Et₂O (150 mL). The mixture was then carefully quenched withsaturated aqueous NaHCO₃ until gas evolution ceased. At this time, theorganic layer was separated and the remaining aqueous layer wasextracted with Et₂O (300 mL). The combined organic layers were washedwith brine (200 mL), dried (Na₂SO₄), filtered, and concentrated invacuo. Purification by silica gel chromatography eluting with 20%-50%EtOAc/hexanes afforded the title compound (0.960 g, 93%): ¹H NMR (300MHz. CDCl₃) δ 7.34-7.30 (m, 5H), 5.11-4.83 (m, 3H), 4.21 (q, J=7.2 Hz,2H), 3.37-3.25 (m, 2H), 1.73-1.68 (m, 1H), 1.27 (t, J=7.2 Hz, 3H),1.14-1.06 (m, 1H); ESI MS m/z 280 [M+H]⁺.

2-(Benzyloxycarbonylamino)-1-hydroxycyclopropanecarboxylic acid (7b)

To a 0° C. solution of ethyl2-(benzyloxycarbonylamino)-1-hydroxycyclopropanecarboxylate (6b, 12.5 g,44.7 mmol) in THF (100 mL) was added K₂CO₃ (24.7 g, 179.0 mmol) as asolution in H₂O (300 mL). The reaction was allowed to warm to roomtemperature and stirred for 4 h and then additional H₂O (200 mL) wasadded. After stirring an additional 18 h at room temperature thereaction was concentrated to remove most of the THF. The remainingaqueous solution was washed with Et₂O (2×500 mL), acidified with 2 N HClto pH 2, and then extracted with EtOAc (5×200 mL). The combined EtOAclayers were washed with brine (500 mL), dried (Na₂SO₄), filtered andconcentrated in vacuo to afford the title compounds (7.75 g, 69%) as amixture of diastereomers. The mixture was triturated with Et₂O to afforda white solid as mostly the major diastereomers. The supernatant wasconcentrated and then triturated with Et₂O to afford a clean mixture ofboth diastereomers. Major Diastereomer: ¹H NMR (300 MHz, MeOD) δ7.50-7.14 (m, 5H), 5.22-4.96 (m, 2H), 3.23-3.10 (m, 1H), 1.60 (dd,J=8.9, 6.3 Hz, 1H), 1.10 (t, J=6.2 Hz, 1H); Multimode (APCI+ESI) MS m/z250 [M−H]⁻. Mixture of Diastereomers: ¹H NMR (300 MHz, MeOD) δ 7.45-7.14(m, 5H), 5.24-5.01 (m, 2H), 3.25-3.15 (m, 0.46H), 3.14-3.01 (m, 0.54H),1.71-1.53 (m, 1H), 1.42 (dd, J=9.1, 6.4 Hz, 0.54H), 1.12 (t, J=6.2 Hz,0.46H); Multimode (APCI+ESI) MS m/z 250 [M−H]⁻.

Representative Compounds

The following representative compounds may be prepared according to theforegoing procedures.

MIC Assay Protocol

Minimum inhibitory concentrations (MIC) are determined by referenceClinical and Laboratory Standards Institute (CLSI) broth microdilutionmethods per M7-A7 [2006]. Quality control ranges utilizing E. coli ATCC25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 29213, andinterpretive criteria for comparator agents are as published in CLSIM100-S17 [2007]. Briefly, serial two-fold dilutions of the testcompounds are prepared at 2× concentration in Mueller Hinton Broth. Thecompound dilutions are mixed in 96-well assay plates in a 1:1 ratio withbacterial inoculum. The inoculum is prepared by suspension of a colonyfrom an agar plate that is prepared the previous day. Bacteria aresuspended in sterile saline and added to each assay plate to obtain afinal concentration of 5×10⁵ CFU/mL. The plates are incubated at 35° C.for 20 hours in ambient air. The MIC is determined to be the lowestconcentration of the test compound that resulted in no visible bacterialgrowth as compared to untreated control.

In Vivo Efficacy Models

Compounds are tested for in vivo efficacy in a murine septicemia modelof infection. Two models are run on each compound, using E. coli and P.aeruginosa QC bacterial strains. Both studies employ the same design.Male CD-1 (CRL)-derived mice (individual body weight, 24±2 grams) areinoculated IP with the 2×LD90-100 dose of E. coli ATCC 25922 (4.5×105CFU/mouse) in 0.5 mL of BHI broth containing 5% mucin, or the 2×LD90-100dose of P. aeruginosa ATCC 27853 (5.8×104 CFU/0.5 mL/mouse) in BHI brothcontaining 5% mucin. At 1 hour after bacterial challenge, the micereceive a single SC or IV dose of vehicle or test substance to assess invivo anti-infective activity. Mortality is recorded once daily for 7days after bacterial inoculation.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A compound having the following structure (I):

or a stereoisomer or pharmaceutically acceptable salt thereof, wherein:Q₁ is

Q₂ is hydrogen, substitute alkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

Q₃ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted cycloalkyl,optionally substituted cycloalkylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—C(═NH)NR₇R₈,

each R₁ and R₂ is, independently, hydrogen or an amino protecting group;each R₃ is, independently, hydrogen or a hydroxyl protecting group; eachR₄, R₅, R₇ and R₈ is, independently, hydrogen or C₁-C₆ alkyl optionallysubstituted with one or more halogen, hydroxyl or amino; each R₆ is,independently, hydrogen, halogen, hydroxyl, amino or C₁-C₆ alkyl; or R₄and R₅ together with the atoms to which they are attached can form aheterocyclic ring having from 4 to 6 ring atoms, or R₅ and one R₆together with the atoms to which they are attached can form aheterocyclic ring having from 3 to 6 ring atoms, or R₄ and one R₆together with the atoms to which they are attached can form acarbocyclic ring having from 3 to 6 ring atoms, or R₇ and R₈ togetherwith the atom to which they are attached can form a heterocyclic ringhaving from 3 to 6 ring atoms; each R₉ is, independently, hydrogen,hydroxyl, amino or C₁-C₆ alkyl optionally substituted with one or morehalogen, hydroxyl or amino; each R₁₀ is, independently, hydrogen,halogen, hydroxyl, amino or C₁-C₆ alkyl; or R₉ and one R₁₀ together withthe atoms to which they are attached can form a heterocyclic ring havingfrom 3 to 6 ring atoms; and n is an integer from 1 to 4, and wherein (i)at least one of Q₂ and Q₃ are other than hydrogen.
 2. A compound ofclaim 1 wherein each R₆ is hydrogen.
 3. A compound of claim 2 wherein Q₁is:


4. A compound of claim 1 wherein Q₂ is other than hydrogen.
 5. Acompound of claim 4 wherein Q₃ is hydrogen.
 6. A compound of claim 4wherein Q₂ is alkyl substituted with hydroxyl or amino.
 7. A compound ofclaim 1 wherein each R₁, R₂ and R₃ is hydrogen.
 8. A pharmaceuticalcomposition comprising a compound of claim 1, or a stereoisomer orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, diluent or excipient.
 9. A method of treating abacterial infection in a mammal in need thereof, comprisingadministering to the mammal an effective amount of a compound of claim 1or a composition of claim 8.